Plant lysophosphatidic acid acyltransferases

ABSTRACT

This invention relates to plant LPAATs, means to identify such proteins, amino acid and nucleic acid sequences associated with such protein, and methods to obtain, make and/or use such plant LPAATs. Purification, especially the removal of plant membranes and the substantial separation away from other plant proteins, and use of the plant LPAAT is provided, including the use of the protein as a tool in gene isolation for biotechnological applications. In addition, nucleic acid sequences encoding LPAAT protein regions are provided, and uses of such sequences for isolation of LPAAT genes from plants and for modification of plant triglyceride compositions are considered.

This application is a continuation-in-part of application Ser. No.08/231,196 filed Apr. 21, 1994 and a continuation-in-part of applicationSer. No. 08/224,625 filed Apr. 6, 1994, now U.S. Pat. No. 5,563,058.

TECHNICAL FIELD

The present invention is directed to protein preparations, amino acidand nucleic acid sequences and constructs, and methods related thereto.

INTRODUCTION Background

There is a need for improved means to obtain or manipulate fatty acidcompositions, from biosynthetic or natural plant sources. For example,novel oil products, improved sources of synthetic triacylglycerols(triglycerides), alternative sources of commercial oils, such astropical oils (i.e., palm kernel and coconut oils), and plant oils foundin trace amounts from natural sources are desired for a variety ofindustrial and food uses.

To this end, the triacylglycerol (TAG) biosynthesis system in plants andbacteria has been studied. In the cytoplasmic membranes of plant seedtissues which accumulate storage triglycerides ("oil"), fatty acylgroups at the sn-2 position of the triglyceride molecules areincorporated via action of the enzyme 1-acylglycerol-3-phosphateacyltransferase (E.C. 2.3.1.51), also known as lysophosphatidic acidacyltransferase, or LPAAT.

By inspection of the LPAAT activities in isolated membranes from seedtissues, it has been shown that LPAAT specificities vary from species tospecies in accordance with the kinds of fatty acyl groups found in thesn-2 positions of the respective storage oils. For example, in the seedsof Cuphea species, which accumulate oils containing medium-chain fattyacids, it is possible to demonstrate an LPAAT activity which willutilize medium-chain acyl-CoA and lysophosphatidic acid (LPA)substrates. In contrast, LPAAT activity from the membranes of rapeseedembryos, in which the oil contains fatty acids of longer chain length,uses these medium-chain substrates much less readily, and predominantlyuses long-chain unsaturated fatty acids. Similarly the meadowfoam plant(Limnanthes alba) accumulates an oil containing erucic acid (22:1) inall three sn positions and has a seed LPAAT activity able to use22:1-CoA and 22:1-LPA, whereas rapeseed, which does not accumulate thesefatty acids, has little or no such 22:1-utilizing LPAAT.

Similar studies with the enzymes responsible for the sn-1 and sn-3acylations show that they are much less selective with respect to thesubstrate chain lengths. Thus, for a specific storage triglyceride in agiven plant, the types of fatty acyl groups found in the sn-2 positionof the oil are determined primarily by the specificity of LPAAT withrespect to its acyl-donor substrates, i.e. acyl-CoAs. In addition, theselectivity of the LPAAT towards the acyl-CoAs is also influenced by thenature of the acyl group already attached in the sn-1 position of theacceptor substrates, i.e. the 1-acylglycerol-3-phosphate orlysophosphatidic acid (LPA) molecules.

The characterization of lysophosphatidic acid acyltransferase (alsoknown as LPAAT) is useful for the further study of plant FAS systems andfor the development of novel and/or alternative oils sources. Studies ofplant mechanisms may provide means to further enhance, control, modifyor otherwise alter the total fatty acyl composition of triglycerides andoils. Furthermore, the elucidation of the factor(s) critical to thenatural production of triglycerides in plants is desired, including thepurification of such factors and the characterization of element(s)and/or co-factors which enhance the efficiency of the system. Of specialinterest are the nucleic acid sequences of genes encoding proteins whichmay be useful for applications in genetic engineering.

LITERATURE

Published characterizations of acyltransferase specificities in rapeseedmembranes report that acyl group discrimination occurs primarily at thesn-2 acylation (Oo et al., Plant Physiol. (1989) 91:1288-1295; Bernerthet al, Plant Sci. (1990) 67:21-28).

Coleman (Mol. Gen. Genet. (1992) 232:295-303) reports thecharacterization of an E. coli gene (plsC) encoding LPAAT. The E. coliLPAAT is capable of utilizing either acyl-CoA or acyl-ACP as the fattyacyl donor substrate.

Hares & Frentzen (Planta (1991) 185:124-131) report solubilization andpartial purification of a long-chain preferring LPAAT from endoplasmicreticulum in pea shoots. The purported solubilization is based solely onthe inability to sediment LPAAT activity by high-speed centrifugation.

Wolter et al. (Fat Sci. Technol. (1991) 93: 288-290) report failedattempts to purify a Limnanthes douglaslii acyltransferase catalyzingthe acylation of erucic acid to the sn-2 position of the glycerolbackbone, and propose hypothetical methods of gene isolation based oncDNA expression in microorganisms.

Nagiec et al. (J. Biol. Chem. (1993) 268:22156-22163) report the cloningof an SLCI (sphingolipid compensation) gene from yeast and reporthomology of the encoded protein to the LPAAT protein of E. coli.

Taylor et al. (in "Seed Oils for the Future", ed. Mackenzie & Taylor(1992) AOCS Press) report acyl-specificities for 18:1-CoA and 22:1-CoAsubstrates for LPAATs from several plant species and discuss attempts topurify a B. napus LPAAT.

Slabas et al. (Ch. 5, pages 81-95 (1993) in Seed Storage Compounds:Biosynthesis, Interactions, and Manipulation, ed Shewry & Stobart,Clarendon Press) discuss attempts to purify plant LPAAT proteins andnote that all attempts to purify LPAAT to homogeneity have failed.Attempts to clone a corn LPAAT gene by complementation of an E. colimutation at plsC are also discussed

Oo et al. (Plant Physiol. (1989) 91:1288-1295) report characterizationof LPAAT specificities in membrane preparations of palm endosperm, maizescutellum, and rapeseed cotyledon.

Cao et al. (Plant Physiol. (1990) 94:1199-1206) report characterizationof LPAAT activity in maturing seeds of meadowfoam, nasturtium, palm,castor, soybean, maize, and rapeseed. LPAAT activity was characterizedwith respect to 22:1 and 18:1 LPA and acyl-COA substrates.

Laurent and Huang (Plant Physiol. (1992) 99:1711-1715) report thatLPAATs in palm and meadowfoam which are capable of transferring 12:0 and22:1 acyl-CoA substrates to the sn-2 position of LPA, are confined tothe oil-accumulating seed tissues.

Bafor et al. (Phytochemistry (1990) 31:2973-2976) report substratespecificities of TAG biosynthesis enzymes, including LPAAT, from Cupheaprocumbens and C. wrighti.

Bafor et al. (Biochem. J. (1990) 272:31-38) report results of studies onregulation of TAG biosynthesis in Cuphea lanceolata embryos. Results ofassays for LPAAT activity in microsomal preparations from developingcotyledons are provided.

Frentzen et al. (Eur. J. Biochem. (1990) 187:389-402 reportcharacterization of mitochondrial LPAAT activity in potato tubers andpea leaves.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of soybean phospholipid concentration on coconutmedium-chain LPAAT activity (assay of S3 preparation).

FIG. 2 shows the results of chromatography of bay P2 preparation onSephacryl S400 column.

FIG. 3 shows the results of a bay supernatant fraction preparedaccording to Frentzen et al., and chromatographed on a Sephacryl S400column.

FIG. 4 shows the results of chromatography of the bay S3 preparation ona Superose 6 column.

FIG. 5 provides a demonstration of the effects of solubilization byCHAPS concentration and detergent/protein (D/P) ratio, as measured bythe yield of coconut medium-chain LPAAT activity in the S3 preparation.

FIG. 6 shows the chromatography of coconut S3 preparation on red 120agarose.

FIG. 7 shows the results of chromatography of coconut medium-chain LPAATactivity from the red 120 column on a column of hydroxyapatite.

FIG. 8 shows the results of partially purified coconut medium-chainLPAAT preparation passed through a 12:0-CoA chromatography column.

FIG. 9 provides the results of chromatography of partially purified,PL-activated coconut medium-chain LPAAT preparation on a 12:0-CoA columnin the presence of phospholipids.

FIG. 10 provides DNA sequence and translated amino acid sequence of aclone, 23-2 (SEQ ID NO:18), containing LPAAT encoding sequence obtainedby PCR.

FIG. 11 provides DNA sequence and translated amino acid sequence of aclone, 23-4 (SEQ ID NO:19), containing LPAAT encoding sequence obtainedby PCR.

FIG. 12 provides DNA sequence and translated amino acid sequence of aclone, 10-1 (SEQ ID NO:20), containing LPAAT encoding sequence obtainedby PCR.

FIGS. 13A-13D provide DNA sequence and translated amino acid sequence offull length coconut LPAAT clone COLP4 (pCGN5503), (SEQ ID NO:21). FIG.13A provides nucleotides 1-435. FIG. 13B provides nucleotides 436-771.FIG. 13C provides nucleotides 772-1059. FIG. 13D provides nucleotides1060-1408.

SUMMARY OF THE INVENTION

This invention relates to plant proteins which catalyze the productionof 1,2-diacylglycerol-3-phosphate from 1-acylglycerol-3-phosphate (alsoreferred to as lysophosphatidic acid or LPA) and an acyl-CoA substrate.Such proteins are referred to herein as 1-acylglycerol-3-phosphateacyltransferases (E.C. 2.3.1.51) or LPAATs. In particular, the LPAATproteins of this invention demonstrate preferential activity on acyl-CoAdonor substrates and little or no activity towards acyl-ACP donorsubstrates.

By this invention, plant LPAAT proteins are substantially purified awayfrom the cytoplasmic membranes of their native plant host, andcharacterized with respect to preferential substrate activity. Inparticular, purification of a plant LPAAT enzyme having preferentialactivity towards medium-chain acyl-CoA substrates is provided.

A medium-chain preferring LPAAT of this invention demonstrates apreference for medium-chain acyl-CoA donor substrates, whether the LPAacceptor substrate contains a medium-chain acyl group (such as C12:0) atthe sn-1 position or a long-chain acyl group (such as C18:1) at the sn-1position. A coconut endosperm medium-chain acyl-CoA preferring LPAATenzyme is exemplified herein. Lauroyl-CoA is a preferred donor substratewhen the acceptor substrate is either 1-lauroylglycerol-3-phosphate or1-oleoylglycerol-3-phosphate. In addition, the coconut LPAAT alsodemonstrates preferential activity on other medium-chain acyl-CoAsubstrates, particularly those having C10 or C14 carbon chains, ascompared to longer chain length (C16 or C18) substrates.

The exemplified coconut LPAAT is purified away from the membranes (i.e.solubilized), and the solubilized LPAAT preparation is subjected tovarious chromatographic analyses to identify a protein associated withthe LPAAT activity. In this manner a protein having a molecular weightof approximately 27-29 kDA is identified as associated with LPAATactivity. Further purification methods, such as column chromatographyand polyacrylamide gel electrophoresis are utilized to obtain the LPAATprotein in sufficient purity for amino acid sequence analysis.

As a result, LPAAT peptide sequences are determined, and an LPAATpeptide fragment having sequence homology to a non-plant LPAAT (E. coliplsC gene product) is discovered. The LPAAT peptide sequences are usedas templates in designing various synthetic oligonucleotides which arethen used to obtain nucleic acid sequences encoding all or a portion ofthe coconut LPAAT protein.

LPAAT PCR product sequences are provided in the instant application andused to obtain cDNA clones encoding coconut LPAAT, sequence of which isalso provided herein. Using the coconut LPAAT encoding sequences soobtained, it is also possible to isolate other plant LPAAT genes whichencode LPAAT proteins of different specificities with respect toacyl-CoA donor substrates (e.g. 8:0, 10:0, 14:0, 22:1 etc.).

Thus, this invention encompasses plant LPAAT peptides and thecorresponding amino acid sequences of those peptides, and the use ofthese peptide sequences in the preparation of oligonucleotidescontaining LPAAT encoding sequences for analysis and recovery of plantLPAAT gene sequences. The plant LPAAT encoding sequence may encode acomplete or partial sequence depending upon the intended use. All or aportion of the genomic sequence, or cDNA sequence, is intended.

Of special interest are recombinant DNA constructs which provide fortranscription or transcription and translation (expression) of the plantLPAAT sequences. In particular, constructs which are capable oftranscription or transcription and translation in plant host cells arepreferred. Such constructs may contain a variety of regulatory regionsincluding transcriptional initiation regions obtained from genespreferentially expressed in plant seed tissue.

In yet a different aspect, this invention relates to a method forproducing a plant LPAAT in a host cell or progeny thereof via theexpression of a construct in the cell. Cells containing a plant LPAAT asa result of the production of the plant LPAAT encoding sequence are alsocontemplated herein.

In addition, this invention relates to methods of using DNA sequencesencoding plant LPAAT for the modification of the proportion fatty acylgroups at the sn-2 position of the triglyceride molecules, especially inthe seed oil of plant oilseed crops. Plant cells having such a modifiedtriglyceride are also contemplated herein. Of particular interest is theuse of a medium-chain preferring LPAAT sequence in Brassica plants whichhave been engineered to produce medium-chain fatty acids in the seedoil. In such plants, up to approximately 50 mol percent laurate isaccumulated in the seed triglycerides. Most of this laurate, however, isesterified at the sn-1 and sn-3 positions due to the specificity of theBrassica LPAAT for longer chain length acyl-CoA substrates. Byexpression of a medium-chain preferring LPAAT protein in the seeds ofsuch plants, it is possible to obtain Brassica seed oil which hasgreater than 67 mole percent laurate in the TAG.

Also considered in this invention are the modified plants, seeds andoils obtained by expression of the plant LPAAT proteins of thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

A plant LPAAT of this invention includes any sequence of amino acids,such as a protein, polypeptide or peptide, obtainable from a plantsource, which demonstrates the ability to catalyze the production of1,2-diacylglycerol-3-phosphate from 1-acylglycerol-3-phosphate and anacyl-CoA substrate under plant enzyme reactive conditions. By "enzymereactive conditions" is meant that any necessary conditions areavailable in an environment (i.e., such factors as temperature, pH, lackof inhibiting substances) which will permit the enzyme to function.

Preferential activity of a plant LPAAT toward particular chain-lengthfatty acyl-CoA substrates is determined upon comparison of1,2-diacylglycerol-3-phosphate product amounts obtained per differentchain length acyl-CoA donor substrates. In some cases, the chain lengthof an acyl group in the sn-1 position may also affect the ability of theLPAAT to utilize a given chain length acyl-CoA donor. Of particularinterest in the instant invention is a medium-chain acyl-CoA preferringLPAAT in coconut immature endosperm tissue.

By medium-chain acyl-CoA preferring is meant that the enzyme preparationdemonstrates a preference for medium-chain, i.e. C8, C10, C12 or C14acyl-CoA donor substrates over acyl-CoA substrates of different acylcarbon lengths, regardless of the chain length of the acyl group in thesn-1 position of the acceptor LPA substrate. It is noted that someactivity, of a lesser magnitude, may also be observed against otherchain-length fatty acyl substrates, i.e., the specificity will besubstantial, but may not be absolute. For example, the exemplifiedcoconut LPAAT demonstrates a strong preference for C12 acyl-CoA donorsubstrates when the acceptor substrate is lauroyl-LPA, but also hassignificantly more activity towards C10 and C14 substrates as comparedto longer chain substrates whose acyl groups have 16 or 18 carbons. Whenthe acceptor substrate is 18:1-LPA, the coconut LPAAT uses C12 and C14substrates at nearly equal rates, and still prefers these and C10substrates over available long-chain acyl-CoA substrates.

Other plant LPAAT proteins may also demonstrate preferential activity onone or more medium-chain acyl-CoA substrates, but the preference mayonly be encountered where a medium-chain acyl group is present in thesn-1 position of the LPA donor substrate. Such LPAAT's are considered ashaving selective preference for medium-chain acyl-CoA substrates.

As noted above, a plant LPAAT of this invention will display activitytoward fatty acyl-CoA substrates, and have little or no activity towardsfatty acyl-ACP substrates. Thus, the LPAAT of the instant invention maybe distinguished from plant chloroplastic LPAATs which demonstrateactivity towards both acyl-ACP and acyl-CoA substrates.

The acyl-CoA LPAATs of the instant invention are present in cytoplasmicmembranes in various plant tissues. Of particular interest are thoseLPAATs associated with the TAG biosynthesis pathway in the endoplasmicreticulum of immature seed tissues. Immature seed tissues containingsuch LPAATs may include embryo tissue or endosperm tissue, depending onthe location of TAG biosynthesis in a particular plant species. Incoconuts, for example, LPAAT activity is detected primarily in theendosperm tissue, the site of TAG biosynthesis. In California bay seeds,immature embryo cotyledons provide a good source of LPAAT activity, andin Brassica seeds, substantial LPAAT activity is also found in immatureembryos.

The plant endoplasmic reticulum LPAAT enzymes studied to date have beenfound to be membrane proteins. Thus, in order to further study LPAATactivity, and in particular to produce purified preparations of such aprotein by chromatographic methods, it is necessary to obtain the enzymein solubilized form, i.e. separated from the cytoplasmic membraneenvironment.

"Solubilization" refers to extraction of the LPAAT enzyme from themembranes in such a way that it then behaves in a manner typical ofenzymes that are not membrane-associated. Because the membraneeffectively links the LPAAT protein to other proteins which are alsopresent therein, solubilization is an essential requirement foridentification and purification of the LPAAT protein as described in thefollowing examples. In testing for solubilization of LPAAT activity,three different indications of solubilization, as described in moredetail in the following examples, are considered.

1) LPAAT activity is not sedimented by very high-speed centrifugation.

2) LPAAT activity migrates on a size-exclusion chromatography column asthough it had a native molecular weight typical of enzymes which are notmembrane-associated.

3) Proteins present in the LPAAT preparation are at least partiallyseparable from each other by column chromatography.

Because of potential alternative interpretations that may apply to anyof the above criteria individually, it is necessary to confirm that allthree of the criteria have been satisfied to confirm LPAATsolubilization. For example, the first criterion, of failure to sedimentat very high g forces could be misleading if the density of the solutionused for solubilization is similar to that of the unsolubilizedmembranes so that they sediment only very slowly. This situation isillustrated in the examples which follow, in which a publishedsolubilization procedure that relied on this criterion alone, is shownto be inadequate to obtain LPAAT substantially separated from thecytoplasmic membranes. The second criterion, in which solubilizedactivity migrates more slowly through a size-exclusion column than theoriginal membranes, may be compromised if the membranes themselves bindweakly to the column after exposure to detergent so that their migrationthrough it is slowed. The third criterion, in which the solubilizedproteins are chromatographically resolvable, is the least likely to becompromised by artifacts or unforeseen situations. However, it ispossible that membranes could be partially dissociated by thesolubilization procedure such that various aggregates of proteins arereleased. Such aggregates might then be resolved from each otherchromatographically. Thus, satisfaction of all three criteria isnecessary to assure that LPAAT solubilization is achieved.

Solubilization of coconut LPAAT in a solution containing 1M NaCl, 2.25%(w/v) CHAPS detergent, and a detergent/protein ratio of 48/1 (w/w) isdescribed in the following examples. Similarly, LPAAT activity fromCalifornia bay is solubilized using a solubilization solution containing1M NaCl, 4% (w/v) CHAPS detergent, and a detergent/protein ratio of 58/1(w/w) Solubilization of the plant LPAATs is confirmed by demonstrationof each of the above criteria of solubilization.

Furthermore, in studies of the solubilized LPAAT activity it wasdiscovered, as described in detail in the following examples, thatsolubilized LPAAT could only be assayed by addition of concentratedphospholipids, to reconstitute LPAAT activity. In particular, thestimulatory action of phospholipids on LPAAT activity is greatest whenthe phospholipids are added to the solubilized LPAAT sample at the startof the assay procedure, followed by dilution of the high CHAPS and saltconcentrations in this buffer by addition of the remaining assayingredients. Addition of the phospholipids after dilution of thesolubilization solution results in little or no increase in detection ofLPAAT activity. The phospholipid stimulation effect is also seen wherethe phospholipids are added to a sample of solubilization buffer alone,followed by dilution with remaining assay ingredients and subsequentaddition of the solubilized LPAAT sample.

Solubilized preparations of coconut endosperm LPAAT are utilized in avariety of chromatographic experiments for identification and partialpurification of the LPAAT protein. In this manner, a protein having amolecular weight of approximately 27-29 kDa is identified as associatedwith LPAAT activity. As described in more detail in the followingexamples, the 29 kDa protein is partially purified by chromatography onred 120 agarose and hydroxyapatite columns. The protein is then obtainedin substantially purified form by gel electrophoresis and blotting ofthe partially purified LPAAT preparation to nitrocellulose. The 27-29kDA protein is recovered by cutting out that portion of thenitrocellulose filter containing the identified band.

The purified protein is then digested with various enzymes to generatepeptides for use in determination of amino acid sequence. Amino acidsequence of a tryptic peptide obtained in this manner is demonstrated toshare a region of homology with the LPAAT protein encoded by the E. coliplsC gene. This same region shared by the E. coli and coconut LPAATs isalso found in a yeast acyltransferase protein encoded by the SLC1 gene.

Thus, the tryptic peptide of the 27-29 kDa protein described hereinrepresents a portion of a medium chain-acyl-CoA preferring coconutLPAAT. Other coconut LPAAT peptides are similarly obtained and the aminoacid sequences provided.

The use of amino acid sequences from LPAAT peptides to obtain nucleicacid sequences which encode coconut LPAAT is described herein. Forexample, synthetic oligonucleotides are prepared which correspond to theLPAAT peptide sequences. The oligonucleotides are used as primers inpolymerase chain reaction (PCR) techniques to obtain partial DNAsequence of LPAAT genes. The partial sequences so obtained are then usedas probes to obtain LPAAT clones from a gene library prepared fromcoconut tissue. As an alternative, where oligonucleotides of lowdegeneracy can be prepared from particular LPAAT peptides, such probesmay be used directly to screen gene libraries for LPAAT gene sequences.In particular, screening of cDNA libraries in phage vectors is useful insuch methods due to lower levels of background hybridization. DNAsequences of LPAAT peptide encoding sequences obtained in this mannerare provided in the application.

A nucleic acid sequence of a plant LPAAT of this invention may be a DNAor RNA sequence, derived from genomic DNA, cDNA, mRNA, or may besynthesized in whole or in part. The gene sequences may be cloned, forexample, by isolating genomic DNA from an appropriate source, andamplifying and cloning the sequence of interest using a polymerase chainreaction (PCR). Alternatively, the gene sequences may be synthesized,either completely or in part, especially where it is desirable toprovide plant-preferred sequences. Thus, all or a portion of the desiredstructural gene (that portion of the gene which encodes the LPAATprotein) may be synthesized using codons preferred by a selected host.Host-preferred codons may be determined, for example, from the codonsused most frequently in the proteins expressed in a desired hostspecies.

One skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared and used to screen and recover "homologous" or "related" LPAATsfrom a variety of plant sources. Homologous sequences are found whenthere is an identity of sequence, which may be determined uponcomparison of sequence information, nucleic acid or amino acid, orthrough hybridization reactions between a known LPAAT and a candidatesource. Conservative changes, such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lysand Gln/Asn may also be considered in determining sequence homology.Amino acid sequences are considered homologous by as little as 25%sequence identity between the two complete mature proteins. (Seegenerally, Doolittle, R. F., OF URFS and ORFS (University Science Books,CA, 1986.)

Thus, other plant LPAATs may be obtained from the specific exemplifiedcoconut protein preparations and sequences provided herein. Furthermore,it will be apparent that one can obtain natural and synthetic plantLPAATs, including modified amino acid sequences and starting materialsfor synthetic-protein modeling from the exemplified plant LPAATs andfrom plant LPAATs which are obtained through the use of such exemplifiedsequences. Modified amino acid sequences include sequences which havebeen mutated, truncated, increased and the like, whether such sequenceswere partially or wholly synthesized. Sequences which are actuallypurified from plant preparations or are identical or encode identicalproteins thereto, regardless of the method used to obtain the protein orsequence, are equally considered naturally derived.

Typically, a plant LPAAT sequence obtainable from the use of nucleicacid probes will show 60-70% sequence identity between the target LPAATsequence and the encoding sequence used as a probe. However, lengthysequences with as little as 50-60% sequence identity may also beobtained. The nucleic acid probes may be a lengthy fragment of thenucleic acid sequence, or may also be a shorter, oligonucleotide probe.When longer nucleic acid fragments are employed as probes (greater thanabout 100 bp), one may screen at lower stringencies in order to obtainsequences from the target sample which have 20-50% deviation (i.e.,50-80% sequence homology) from the sequences used as probe.Oligonucleotide probes can be considerably shorter than the entirenucleic acid sequence encoding an LPAAT enzyme, but should be at leastabout 10, preferably at least about 15, and more preferably at leastabout 20 nucleotides. A higher degree of sequence identity is desiredwhen shorter regions are used as opposed to longer regions. It may thusbe desirable to identify regions of highly conserved amino acid sequenceto design oligonucleotide probes for detecting and recovering otherrelated LPAAT genes. Shorter probes are often particularly useful forpolymerase chain reactions (PCR), especially when highly conservedsequences can be identified. (See, Gould, et al., PNAS USA (1989)86:1934-1938.)

In addition to isolation of other plant LPAATs, it is considered thatgenes for other related acyltransferase proteins may also be obtainedusing sequence information from the coconut LPAAT and related nucleicacid sequences. For example, other acyltransferase enzymes are involvedin plant lipid biosynthesis, including plastidial LPAAT, mitochondrialLPAAT, lysophosphosphatidylcholine acyltransferase (LPCAT),lysophosphosphatidylserine acyltransferase (LPSAT),lysophosphosphatidylethanolamine acyltransferase (LPEAT), andlysophosphosphatidylinositol acyltransferase (LPIAT). These enzymes allcatalyze acyltransferase reactions involving the sn-2 position oflysophospholipids, and the genes encoding these sequences may also berelated to the plant acyl-CoA LPAAT sequences of the instant inventionand obtainable therefrom.

To determine if a related gene may be isolated by hybridization with agiven sequence, the sequence is labeled to allow detection, typicallyusing radioactivity, although other methods are available. The labeledprobe is added to a hybridization solution, and incubated with filterscontaining the desired nucleic acids, such as Northern or Southernblots, or the filters containing cDNA or genomic clones to be screened.Hybridization and washing conditions may be varied to optimize thehybridization of the probe to the sequences of interest. Lowertemperatures and higher salt concentrations allow for hybridization ofmore distantly related sequences (low stringency). If backgroundhybridization is a problem under low stringency conditions, thetemperature can be raised either in the hybridization or washing stepsand/or salt content lowered to improve detection of the specifichybridizing sequence. Hybridization and washing temperatures can beadjusted based on the estimated melting temperature of the probe asdiscussed in Beltz, et al. (Methods in Enzymology (1983) 100:266-285).In particular, such screening methods may be used to screen mRNApreparations from seed tissues of a variety of plant species to identifyrelated LPAAT or other acyl transferase genes which may be isolatedusing LPAAT gene sequences as probes. A useful probe and appropriatehybridization and washing conditions having been identified as describedabove, cDNA or genomic libraries are screened using the labeledsequences and additional plant LPAAT genes are obtained.

For immunological screening, antibodies to the coconut LPAAT protein canbe prepared by injecting rabbits or mice with the purified protein, suchmethods of preparing antibodies being well known to those in the art.Either monoclonal or polyclonal antibodies can be produced, althoughtypically polyclonal antibodies are more useful for gene isolation.Western analysis may be conducted to determine that a related protein ispresent in a crude extract of the desired plant species, as determinedby cross-reaction with the antibodies to the coconut LPAAT. Whencross-reactivity is observed, genes encoding the related proteins areisolated by screening expression libraries representing the desiredplant species. Expression libraries can be constructed in a variety ofcommercially available vectors, including lambda gt11, as described inManiatis, et al. (Molecular Cloning: A Laboratory Manual, Second Edition(1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

All plants utilize LPAAT proteins in production of membranephospholipids, and thus any given plant species can be considered as asource of additional LPAAT proteins. Plants having significantmedium-chain fatty acids in their seed oils are preferred candidates toobtain plant LPAATs capable of incorporating medium-chain fatty acidsinto the sn-2 position of TAG. Several species in the genus Cupheaaccumulate triglycerides containing medium-chain fatty acids in theirseeds, e.g., procumbens, lutea, hookeriana, hyssopifolia, wrightii andinflata. Another natural plant source of medium-chain fatty acids areseeds of the Lauraceae family. In addition to the exemplified CaliforniaBay (Umbellularia californica), Pisa (Actinodophne hookeri), Sweet Bay(Laurus nobilis) and Cinnamomum camphora (camphor) accumulatemedium-chain fatty acids. Other plant sources include Ulmaceae (elm),Palmae, Myristicaceae, Simarubaceae, Vochysiaceae, and Salvadoraceae.

Also of particular interest are LPAATs from plant species whichincorporate unusual long-chain fatty acids in the storage TAG. Forexample nasturtium and meadowfoam contain 22:1 acyl groups in the seedTAG, and meadowfoam has been shown to contain an LPAAT capable ofincorporating 22:1 (erucic) fatty acyl groups into the sn-2 position. AnLPAAT having such activity may find use in production of "tri-erucic"Brassica oil, which to date is not found due to the selectivity ofBrassica seed LPAAT towards unsaturated fatty acids, such as 18:1 and18:2.

In addition, LPAAT enzymes from plants which contain other unusual fattyacids are of interest and may find use for production of TAG containingthese unusual fatty acids in various plant species. Of interest in thisregard are LPAATs involved in the production of acetylenic fatty acids,such as crepenynic acid from Crepis foetida; fatty acids withcyclopentene substituents, such as gorlic acid from species of thefamily Flacourtiaceae; cyclopropane fatty acids, such as vernolic acidfrom Vernonia galamensis; hydroxylated fatty acids, such as ricinoleicacid from Ricinus communis; furan-containing fatty acids, such as fromExocarpus cupressiformis; fatty acids with several unusual functionalgroups, such as those from Sapium sebiferum, which contain multipledouble bonds and an internal ester function; fatty acids with unusualdouble-bond placement, such as petroselinic acid from some species ofUmbelliferae, Araliaceae, and Garryaceae; and medium-chain fatty acidscontaining double bonds, such as from Lindera species.

It should also be noted that plant LPAATs from a variety of sources canbe used to investigate TAG biosynthesis events of plant lipidbiosynthesis in a wide variety of in vivo applications. Because allplants appear to synthesize lipids via a common metabolic pathway, thestudy and/or application of one plant LPAAT to a heterologous plant hostmay be readily achieved in a variety of species. In other applications,a plant LPAAT can be used outside the native plant source of the LPAATto enhance the production and/or modify the composition of the TAGproduced or synthesized in vitro.

In addition, it is now found that LPAAT from E. coli and the coconutLPAAT have regions of conserved amino acid sequence, which regions arealso conserved in a putative LPAAT protein from yeast. Thus, it may bepossible to design probes from such conserved regions to isolate LPAATencoding sequences from other organisms, such as from animals. SuchLPAAT encoding sequences may also find use in applications describedherein, in particular, in plant genetic engineering techniques forproduction of TAG having particular fatty acyl groups at the sn-2position. For example, an animal LPAAT may find applications in plantgenetic engineering to produce oils having long-chain saturated fattyacyl groups, such as 18:0 in the sn-2 position to provide a source ofuseful TAG for infant formula.

The nucleic acid sequences associated with plant LPAAT proteins willfind many uses. For example, recombinant constructs can be preparedwhich can be used as probes, or which will provide for expression of theLPAAT protein in host cells to produce a ready source of the enzymeand/or to modify the composition of triglycerides found therein. Otheruseful applications may be found when the host cell is a plant hostcell, either in vitro or in vivo. For example, by increasing the amountof a respective medium-chain preferring LPAAT available to the plant TAGbiosynthesis pathway, an increased percentage of medium-chain fattyacids may be obtained in the TAG. In a like manner, for someapplications it may be desired to decrease the amount of LPAATendogenously expressed in a plant cell by anti-sense technology. Forexample, to allow for more opportunity for an inserted foreign LPAAT totransfer medium-chain or unusual longer-chain fatty acyl groups to thesn-2 position, decreased expression of a native Brassica long-chainpreferring LPAAT may be desired.

Thus, depending upon the intended use, the constructs may contain thesequence which encodes the entire LPAAT protein, or a portion thereof.For example, where antisense inhibition of a given LPAAT protein isdesired, the entire LPAAT sequence is not required. Furthermore, whereLPAAT constructs are intended for use as probes, it may be advantageousto prepare constructs containing only a particular portion of an LPAATencoding sequence, for example a sequence which is discovered to encodea highly conserved LPAAT region.

As discussed above, nucleic acid sequence encoding a plant LPAAT of thisinvention may include genomic, cDNA or mRNA sequence. By "encoding" ismeant that the sequence corresponds to a particular amino acid sequenceeither in a sense or anti-sense orientation. By "extrachromosomal" ismeant that the sequence is outside of the plant genome of which it isnaturally associated. By "recombinant" is meant that the sequencecontains a genetically engineered modification through manipulation viamutagenesis, restriction enzymes, and the like.

A cDNA sequence may or may not contain pre-processing sequences, such astransit peptide sequences or targetting sequences to facilitate deliveryof the LPAAT protein (such as mitochondrial LPAAT) to a given organelleor membrane location. The use of any such precursor LPAAT DNA sequencesis preferred for uses in plant cell expression. A genomic LPAAT sequencemay contain the transcription and translation initiation regions,introns, and/or transcript termination regions of the plant LPAAT, whichsequences may be used in a variety of DNA constructs, with or withoutthe LPAAT structural gene. Thus, nucleic acid sequences corresponding tothe plant LPAAT of this invention may also provide signal sequencesuseful to direct protein delivery into a particular organellar ormembrane location, 5' upstream non-coding regulatory regions (promoters)having useful tissue and timing profiles, 3' downstream non-codingregulatory region useful as transcriptional and translational regulatoryregions and may lend insight into other features of the gene.

Once the desired plant LPAAT nucleic acid sequence is obtained, it maybe manipulated in a variety of ways. Where the sequence involvesnon-coding flanking regions, the flanking regions may be subjected toresection, mutagenesis, etc. Thus, transitions, transversions,deletions, and insertions may be performed on the naturally occurringsequence. In addition, all or part of the sequence may be synthesized.In the structural gene, one or more codons may be modified to providefor a modified amino acid sequence, or one or more codon mutations maybe introduced to provide for a convenient restriction site or otherpurpose involved with construction or expression. The structural genemay be further modified by employing synthetic adapters, linkers tointroduce one or more convenient restriction sites, or the like.

The nucleic acid or amino acid sequences encoding a plant LPAAT of thisinvention may be combined with other non-native, or "heterologous",sequences in a variety of ways. By "heterologous" sequences is meant anysequence which is not naturally found joined to the plant LPAAT,including, for example, combinations of nucleic acid sequences from thesame plant which are not naturally found joined together.

The DNA sequence encoding a plant LPAAT of this invention may beemployed in conjunction with all or part of the gene sequences normallyassociated with the LPAAT. In its component parts, a DNA sequenceencoding LPAAT is combined in a DNA construct having, in the 5' to 3'direction of transcription, a transcription initiation control regioncapable of promoting transcription and translation in a host cell, theDNA sequence encoding plant LPAAT and a transcription and translationtermination region.

Potential host cells include both prokaryotic and eukaryotic cells. Ahost cell may be unicellular or found in a multicellar differentiated orundifferentiated organism depending upon the intended use. Cells of thisinvention may be distinguished by having a plant LPAAT foreign to thewild-type cell present therein, for example, by having a recombinantnucleic acid construct encoding a plant LPAAT therein.

Depending upon the host, the regulatory regions will vary, includingregions from viral, plasmid or chromosomal genes, or the like. Forexpression in prokaryotic or eukaryotic microorganisms, particularlyunicellular hosts, a wide variety of constitutive or regulatablepromoters may be employed. Expression in a microorganism can provide aready source of the plant enzyme. Among transcriptional initiationregions which have been described are regions from bacterial and yeasthosts, such as E. coli, B. subtilis, Sacchromyces cerevisiae, includinggenes such as beta-galactosidase, T7 polymerase, tryptophan E and thelike.

For the most part, the constructs will involve regulatory regionsfunctional in plants which provide for modified production of plantLPAAT, and possibly, modification of the fatty acid composition. Theopen reading frame, coding for the plant LPAAT or functional fragmentthereof will be joined at its 5' end to a transcription initiationregulatory region. In embodiments wherein the expression of the LPAATprotein is desired in a plant host, the use of all or part of thecomplete plant LPAAT gene is desired; namely all or part of the 5'upstream non-coding regions (promoter) together with the structural genesequence and 3' downstream non-coding regions may be employed.

If a different promoter is desired, such as a promoter native to theplant host of interest or a modified promoter, i.e., havingtranscription initiation regions derived from one gene source andtranslation initiation regions derived from a different gene source,numerous transcription initiation regions are available which providefor a wide variety of constitutive or regulatable, e.g., inducible,transcription of the structural gene functions. Thetranscription/translation initiation regions corresponding to suchstructural genes are found immediately 5' upstream to the respectivestart codons. Among transcriptional initiation regions used for plantsare such regions associated with the T-DNA structural genes such as fornopaline and mannopine synthases, the 19S and 35S promoters from CaMV,and the 5' upstream regions from other plant genes such as napin, ACP,SSU, PG, zein, phaseolin E, and the like. Enhanced promoters, such asdouble 35S, are also available for expression of LPAAT sequences. Forsuch applications when 5' upstream non-coding regions are obtained fromother genes regulated during seed maturation, those preferentiallyexpressed in plant embryo tissue, such as ACP and napin-derivedtranscription initiation control regions, are desired. Such"seed-specific promoters" may be obtained and used in accordance withthe teachings of U.S. Ser. No. 07/147,781, filed Jan. 25, 1988 (now U.S.Ser. No. 07/550,804, filed Jul. 9, 1990), and U.S. Ser. No. 07/494,722filed on or about Mar. 16, 1990 having a title "Novel SequencesPreferentially Expressed In Early Seed Development and Methods RelatedThereto," which references are hereby incorporated by reference.Transcription initiation regions which are preferentially expressed inseed tissue, i.e., which are undetectable in other plant parts, areconsidered desirable for TAG modifications in order to minimize anydisruptive or adverse effects of the gene product.

Regulatory transcript termination regions may be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding the plant LPAAT or a convenienttranscription termination region derived from a different gene source,for example, the transcript termination region which is naturallyassociated with the transcript initiation region. Where the transcripttermination region is from a different gene source, it will contain atleast about 0.5 kb, preferably about 1-3 kb of sequence 3' to thestructural gene from which the termination region is derived.

Plant expression or transcription constructs having a plant LPAAT as theDNA sequence of interest for increased or decreased expression thereofmay be employed with a wide variety of plant life, particularly, plantlife involved in the production of vegetable oils for edible andindustrial uses. Most especially preferred are temperate oilseed crops.Plants of interest include, but are not limited to, rapeseed (Canola andHigh Erucic Acid varieties), sunflower, safflower, cotton, soybean,peanut, coconut and oil palms, and corn. Depending on the method forintroducing the recombinant constructs into the host cell, other DNAsequences may be required. Importantly, this invention is applicable todicotyledons and monocotyledons species alike and will be readilyapplicable to new and/or improved transformation and regulationtechniques.

Of particular interest, is the use of plant LPAAT constructs in plantswhich have been genetically engineered to produce a particular fattyacid in the plant seed oil, where TAG in the seeds of nonengineeredplants of the engineered species, do not naturally contain thatparticular fatty acid. For example, in Brassica plants which have beengenetically engineered to produce the medium-chain fatty acids, and inparticular laurate (12:0), in the seed oil, a deficiency in sn-2acylation has been discovered. (See WO 92/20236.) For example, in oilfrom plants in which 40% of the seed oil fatty acyl groups have beenchanged from the long-chain (primarily 18:1) type to 12:0, the 12:0enrichment at the sn-1 and sn-3 positions (averaged together) isapproximately 50% and the 12:0-enrichment at the sn-2 position isapproximately 12%. Additionally, after separation of the intacttriglyceride species by reverse-phase HPLC, it was estimated that only1% of the triglyceride molecules are tri-12:0, whereas the statisticallypredicted proportion from random acylation at all three sn positionswould be 7%. Thus, the expression of a lauroyl-CoA preferring plantLPAAT in such C12 producing Brassica plants is desirable for enhancedincorporation of 12:0 fatty acyl groups into the sn-2 position.

The coconut medium-chain preferring LPAAT may thus be used for enhancingthe incorporation of laurate into storage oil in rapeseed. In addition,production of TAG containing other medium-chain fatty acyl groups inBrassica and other oilseed crop plants is also desired. (See, forexample, WO 92/20236 and WO 94/10288). As the coconut LPAAT hassignificant ability to utilize other medium chain lengths, particularlyC10 and C14, it also has the potential to enhance the incorporation ofthese fatty acids into plant TAG. Furthermore, TAGs having shorter chainfatty acyl groups in all three sn positions are desirable for variousmedical applications. Such TAG molecules may be obtained by expressionof appropriate acyl-ACP thioesterase and LPAAT genes in oilseed cropplants.

Likewise, the expression of any LPAAT which is capable of transferring amedium-chain fatty acyl group into the sn-2 position of an LPA substrateis also desired for applications in crop species engineered to containmedium-chain fatty acids. Preferential activity is not required, so longas the capability of medium-chain utilization is present.

Further plant genetic engineering applications for LPAAT proteins ofthis invention include their use in preparation of structured plantlipids which contain TAG molecules having desirable fatty acyl groupsincorporated into particular positions on the TAG molecules. Forexample, in Brassica plants, the sn-2 position of TAG contains mainlyunsaturated fatty acyl groups. In certain applications, it may bedesirable to have saturated fatty acids at the sn-2 position, and thusan LPAAT from a different plant source may be identified as havingactivity on, for example 16:0 or 18:0 acyl-CoA substrates, and used fortransformation of Brassica.

In addition, in Brassica plants which contain high levels of erucic acid(22:1) in their seed oils (high erucic acid rapeseed or HEAR), little orno 22:1 is found in the sn-2 position of the TAG molecules. A"tri-erucic" HEAR plant having 22:1 in all three of the TAG sn positionsis desirable. Such a seed oil might be obtained for example byexpression of a C22:1 active LPAAT in HEAR plants. A gene encoding suchan LPAAT could be obtained from a plant, such as meadowfoam (Limnanthesalba), whose seeds accumulate oil containing erucic acid (22:1) in allthree sn positions.

The method of transformation in obtaining such transgenic plants is notcritical to the instant invention, and various methods of planttransformation are currently available. Furthermore, as newer methodsbecome available to transform crops, they may also be directly appliedhereunder. For example, many plant species naturally susceptible toAgrobacterium infection may be successfully transformed via tripartiteor binary vector methods of Agrobacterium mediated transformation. Inmany instances, it will be desirable to have the construct bordered onone or both sides by T-DNA, particularly having the left and rightborders, more particularly the right border. This is particularly usefulwhen the construct uses A. tumefaciens or A. rhizogenes as a mode fortransformation, although the T-DNA borders may find use with other modesof transformation. In addition, techniques of microinjection, DNAparticle bombardment, and electroporation have been developed whichallow for the transformation of various monocot and dicot plant species.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin,etc., complementation providing prototrophy to an auxotrophic host,viral immunity or the like. Depending upon the number of different hostspecies the expression construct or components thereof are introduced,one or more markers may be employed, where different conditions forselection are used for the different hosts.

Where Agrobacterium is used for plant cell transformation, a vector maybe used which may be introduced into the Agrobacterium host forhomologous recombination with T-DNA or the Ti- or Ri-plasmid present inthe Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA forrecombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall formation), the latter beingpermissible, so long as the vir genes are present in the transformedAgrobacterium host. The armed plasmid can give a mixture of normal plantcells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coli and Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta,et al., (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0120 515, which are incorporated herein by reference. Alternatively, onemay insert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli, and the other in Agrobacterium. See, for example,McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein thepRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of the plantexpression vectors in host Agrobacterium cells.

Included with the expression construct and the T-DNA will be one or moremarkers, which allow for selection of transformed Agrobacterium andtransformed plant cells. A number of markers have been developed for usewith plant cells, such as resistance to chloramphenicol, kanamycin, theaminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this invention, one or another marker beingpreferred depending on the particular host and the manner ofconstruction.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime for transformation, the bacteria killed, and the plant cellscultured in an appropriate selective medium. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be grown to seedand the seed used to establish repetitive generations and for isolationof vegetable oils.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES Example 1

Assay for LPAAT Activity

A. Assay for LPAAT Activity in Cell-free Homogenates and MembranePreparations

To assay for LPAAT activity, the sample is incubated withlysophosphatidic acid (LPA) and acyl-coenzyme A (acyl-CoA) substrates inbuffered solution. The acyl substituents of the two substrates arechosen to correspond with the specificity of the enzyme being measured.For example, to measure activity of an LPAAT having preference formedium-chain substrates, lauroyl-LPA (lauroyl-lysophosphatidic acid) andlauroyl-CoA may be used, and to measure activity of an LPAAT preferringlonger-chain acyl groups, oleoyl-LPA and oleoyl-CoA may be used. Theacyl group of one substrate is radioactively labeled in order to detectthe product formed. In the examples which follow the acyl substituent ofthe acyl-CoA substrate is radiolabeled with ¹⁴ C in the carboxyl group.LPAAT activity results in transfer of this acyl group from the acyl-CoA"donor" substrate to the LPA "acceptor" substrate, converting the latterinto the product, phosphatidic acid (PA). LPAAT activity is measured asthe amount of radioactive product formed in a given assay time. The PAproduct is radioactive as a result of the transferred radiolabeled acylgroup at the central carbon atom of the molecule, and the quantity of PAformed may be determined by measuring radioactivity of the PA fraction.For this measurement, the PA is first separated from the acyl-CoAsubstrate by solvent partitioning, or by thin-layer chromatography(TLC).

Acyl 1-¹⁴ C!-CoA substrates can be purchased from commercial suppliers,such as Amersham (Arlington Heights, Ill.). Acyl 1-¹⁴ C!-CoA substrateswhich cannot be purchased from commercial suppliers (e.g. lauroyl 1-¹⁴C!-CoA) may be synthesized enzymatically using the method of Taylor etal. (Analyt. Biochem. (1990) 184:311-316). The 1-¹⁴ C!fatty acids usedin the synthesis typically have specific radioactivities of 20 Ci/mol.The radiolabeled acyl-CoA substrate is diluted before use to 12.5 μM andstored in 3 mM sodium acetate (pH 4.8). Oleoyl-LPA is obtained fromcommercial suppliers, and lauroyl-LPA is enzymatically synthesized usingthe method of Ichihara et al. (Eur. J. Biochem. (1987) 167:339-3457),based on the use of phospholipase D to cleave choline from commerciallyavailable lauroyl-lysophosphatidylcholine.

20 μl of the sample to be assayed for LPAAT activity is mixed with 217.5μl of an assay ingredient mixture in a 4-ml, screw-cap vial. Thecomponents of this mixture are adjusted such that after substrateaddition as described below, the final 250 μl assay system will contain:100 mM HEPES-NaOH (pH 7.5) (HEPES=N- 2-hydroxyethyl!piperazine-N'2-ethanesulfonic acid!, 200 mM NaCl, 4% glycerol (v/v), 10 mM EDTA(ethylenediaminetetra-acetate, disodium salt), 5 mM β-ME(β-mercaptoethanol). The LPA substrate is then added (2.5 μl) to providea final concentration of 20 μM. Control samples to determinenonenzymatic background "activity" can be prepared by omitting the LPAATsample or the LPA. The assay incubation is started by addition of 10 μlof the 12.5 mM radiolabeled acyl-CoA solution so that the finalconcentration is 5 μM. If acyl-CoA concentrations vary slightly from12.5 mM the 10 μl volume is changed accordingly to achieve 5 μM finalconcentration, and the volume change accommodated by adjusting the watercontent of the assay mixture so that the total volume and allconcentrations remain unchanged. The incubation takes place in a waterbath at 30° C., for 20-30 minutes.

To stop the assay, 0.25 ml of 1M KCl in 0.2M H₃ P0₄ is added to thevial. At this point, 40 μl BSA (bovine serum albumin, fraction V) at 1mg/ml are added, followed by 0.75 ml of a solution of 67 μg/ml unlabeledPA (acting as a "carrier" to facilitate partitioning) inchloroform/methanol (2:1, v/v). The chain lengths of the PA acyl groupsare chosen to correspond to those used in the assay substrates. Uponthorough mixing of these components the radiolabeled PA product of theLPAAT reaction partitions into the organic phase and away from theunreacted acyl-CoA and LPA. The vial is centrifuged briefly at low speedto facilitate the separation of organic (lower) and aqueous (upper)phases. The aqueous phase is then removed and discarded. The totalradioactivity extracted into the organic phase is determined by liquidscintillation counting; a 100 μl sample of the organic phase istransferred to a 20 ml scintillation vial and allowed to dry, andscintillation fluid (3-5 ml) is added to the vial. The radioactivity ofthe sample, after subtraction of the "minus-enzyme" or "minus-LPA"radioactivities, is taken as an approximate indication of the amount ofPA formed in the LPAAT-catalyzed reaction and therefore of LPAATactivity.

The determination is an approximation due to the presence of non-PAradioactivity in the organic extract. The non-PA radioactivity resultsfrom the partitioning of a small amount of the radiolabeled acyl-CoAsubstrate into the organic layer along with certain impurities in theacyl-CoA (deriving from impurities in the original radioactive fattyacid used in its preparation), and any free fatty acid resulting fromacyl-CoA hydrolysis that may take place.

A more accurate estimation of the LPAAT activity may be obtained byseparating the PA product from these contaminants by TLC. The remainingorganic phase is applied to a silica TLC plate. Ascending chromatographyis carried out for 50 minutes, using the solvent mixturechloroform/pyridine/88% formic acid (50:30:7, v/v). After the plate hasdried, the distribution of radioactivity is visualized and quantitatedusing an AMBIS radioanalytic imaging system (AMBIS Systems Inc., SanDiego, Calif.). From prior application of standard lipid components theRf of the PA is known. The radioactivity associated with the PA spot isexpressed as a percentage of the total radioactivity of the assay sampleloaded on the plate. This ratio provides an indication of the proportionof the scintillation counts which represent the PA product, and may beused to correct the counts to obtain the total PA radioactivity formedin the assay.

For a given LPAAT enzyme source, the effects of incubation time andsample concentration on LPAAT activity are determined to define theconditions under which the assay results (PA radioactivity) provide alinear measure of LPAAT activity. Subsequent assays are then conductedwithin the determined limits.

B. Assay for LPAAT Activity Following Solubilization

After solubilization of LPAAT protein from plant membranes as describedbelow, modification of the above assay conditions is required in orderto detect maximum LPAAT activity. This is especially important after thesolubilized LPAAT has been chromatographed on at least one column. Theimportant modification to the assay is the addition, at the start of theassay procedure, of 1 μl of a concentrated phospholipid (PL) solution to20 μl of the LPAAT-containing sample in a glass vial. The highconcentrations of CHAPS (at least 1% w/v) and NaCl (typically 0.5M orgreater) in the solubilized LPAAT preparation aid in dispersal of thephospholipids. The phospholipid solution is obtained by sonicating crudesoybean phospholipids (L-phosphatidylcholine from soybean, "Type IVs"obtained from Sigma Chemical Company, St. Louis) at 50 mg/ml in 0.5%(w/v) CHAPS (3-(3-cholamidopropyl)-dimethylammonio!-1-propane-sulfonate) until auniform suspension is obtained. Synthetic phospholipids (phosphatidylcholine, inositol, or ethanolamine alone or in combination), and turkeyegg yolk phospholipid preparation, do not offer significant improvementover the crude soybean material.

The remaining assay ingredients (as described above), with the exceptionof the acyl-CoA substrate, are then added as 219 μl of a mixture. Bythis addition, the CHAPS and NaCl are diluted to levels which do nothinder enzyme activity, but the solution does not turn cloudy, whichsuggests that the phospholipids remain dispersed. Radiolabeled acyl-CoA(10 μl, or an appropriately adjusted volume as indicated above) is addedto start the LPAAT-catalyzed reaction and the rest of the assayprocedure is completed as described above.

The effect of the timing of addition of phospholipids in the assaydescribed above is illustrated in the following table:

    ______________________________________                                        Stage of PL addition                                                                          LPAAT Activity (cpm)                                          ______________________________________                                        At start of assay (control)                                                                   914                                                           None added       0                                                            At start of incubation                                                                        231                                                           At end of incubation                                                                           0                                                            ______________________________________                                    

These results demonstrate that the stimulatory action of thephospholipids is greatest when they are added to the LPAAT preparationat the start of the assay procedure, prior to dilution of the CHAPS andNaCl concentrations by addition of the other assay ingredients. Additionof phospholipids after this dilution, or just prior to the addition ofpartitioning mixture (chloroform/methanol etc.), is less effective orineffective.

To determine whether this sequence of phospholipid addition is moreimportant for the LPAAT enzyme or for the phospholipids, a secondexperiment is conducted in which a purified LPAAT preparation (S3preparation that has been purified sequentially on red 120 agarose andhydroxyapatite columns, Example 5 below) is added just prior to thestart of the incubation. In this experiment, the phospholipids are firstmixed with Solubilization Buffer and subsequently diluted with the assaycomponents prior to addition of LPAAT activity.

The results demonstrate that the activity obtained by adding the LPAATpreparation just prior to incubation is identical to that obtained whenthe phospholipids are added at the start of the assay. It is thereforethe treatment of the phospholipids, in exposing them to high CHAPS andNaCl concentrations and then diluting the mixture, that is critical inorder to obtain their activation of LPAAT. The final LPAAT activitydepends on the phospholipid concentration used, increasing up to 20 μgphospholipid/assay and remaining unchanged from 20 to 50 μgphospholipid/assay. This dependence on phospholipid concentration isindependent of S3 concentration. These observations are summarized inFIG. 1.

In the following examples, where solubilized and column-chromatographedcoconut LPAAT preparations are implicated, the assay data refer to thismodified assay method involving the use of soybean phospholipids.

It is not possible to activate the solubilized bay long-chain LPAAT inthis way to obtain maximal activity; when the phospholipids are includedin the bay assay an alternative reaction occurs, diverting theradiolabeled acyl group from the 18:1-CoA to another productdistinguishable from the LPAAT product (PA) by TLC.

Example 2

Preparation of Cell-free Homogenates and Membrane Fractions with LPAATActivity

A. Coconut LPAAT

Coconuts (Cocos nucifera) are obtained from local supermarket stores.For maximum yield of LPAAT activity, immature coconuts referred to as"green", which have a very pale brown or white endocarp (exterior"shell") are used. The endocarp of the coconut is pierced and the "milk"liquid within the hollow interior drained and discarded. The coconut isthen broken into fragments so that the white endosperm tissue lining theinside of the endocarp can be dissected and collected. The brown testabetween the endosperm and the endocarp is removed and discarded, and theendosperm is frozen by immersion in liquid nitrogen and stored at -70°C. for future use. In a typical preparation as described below, 24 g oftissue are processed. As individual coconuts may vary considerably withrespect to the maturity of the endosperm and therefore the yield ofobtainable LPAAT, the endosperm may be sampled to assess the LPAATcontent prior to beginning a 24 g-scale preparation. Such a sampling maybe accomplished by cutting a hole in the endocarp, approximately 1 inchin diameter. The resulting disc of endosperm is dissected away from thetesta and endocarp and processed as described below except that 16 mlExtraction Buffer are used for analysis of a 2 g powdered endospermsample.

Frozen coconut endosperm tissue is powdered by impact crushing in asteel mortar and pestle in liquid nitrogen. The powder from 24 g oftissue is added to 144 ml Extraction Buffer at 0°-4° C., and the mixtureis blended with a Polytron tissue homogenizer to make a cell-freehomogenate. Extraction Buffer contains 50 mM HEPES-NaOH (pH 7.5), 3MNaCl, 10 mM EDTA, 10 mM DIECA (diethyldithiocarbamic acid, sodium salt),100 μM Pefabloc (protease inhibitor available from Sigma Chemical Co.),1 μM leupeptin, 0.1 μM pepstatin A, 5 mM β-ME. All subsequent steps areperformed at 4° C.

The homogenate is filtered through 4 layers of cheesecloth which hasbeen wetted with Extraction Buffer. The remaining solids are enfolded inthe cheesecloth and the cheesecloth wrung to extract more liquid. Thecheesecloth is then unfolded, the solids wetted with 48 ml of ExtractionBuffer, and the cheesecloth wrung again. The resulting filtrate iscentrifuged at 12,000×g for 30 minutes. The resulting sample contains afloating fat pad and a pellet, which are both discarded, and asupernatant fraction (S1). The supernatant fraction is filtered toremove residual solids using Miracloth (Calbiochem; La Jolla, Calif.)which has been wetted with Extraction Buffer. This S1 fraction is thendialyzed overnight against 4 liters of Dialysis Buffer (50 mM HEPES-NaOHpH 7.5, 1M NaCl, 5 mM β-ME), with one change of buffer. Dialysismembrane having a molecular weight cutoff of 12,000-14,000 is used. Thedialyzed S1 material (DS1) is then centrifuged at 12,000×g for 30minutes and the supernatant fraction again filtered throughbuffer-wetted Miracloth.

The DS1 supernatant is then centrifuged at 100,000×g for 2 hours. Theresulting sample contains a pelleted fraction containing subcellularmembranes (P2), and a supernatant fraction which is discarded. Residualsupernatant fraction is removed from the P2 fraction by draining thecentrifuge tubes and wiping with paper tissues.

P2 Buffer (100 mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 20% glycerol (w/v),10 mM EDTA, 5 mM β-ME) is added to the P2 pellets so that when themixture is transferred to a ground glass homogenizer and homogenized,the total volume of the homogenate will be 2.5 ml. The P2 homogenate isdivided into aliquots, frozen in liquid nitrogen, and stored at -70° C.for future use.

B. California bay LPAAT

A P2 membrane homogenate from immature cotyledons of developingCalifornia bay (Umbellularia californica) seeds is prepared essentiallyas described above, except as noted below. The seeds are dissected, andthe pale green cotyledons are removed, frozen in liquid nitrogen andstored at -70° C. The frozen bay tissue is powdered in liquid nitrogenas described above. Typically 20 g of powdered embryo tissue arehomogenized with Modified Extraction Buffer (100 mM HEPES-NaOH pH 7.5,3M NaCl, 10 mM DIECA, 100 μM PMSF (phenylmethylsulfonyl fluoride), 1 μMleupeptin, 0.1 μM pepstatin A) in a final volume of 200 ml. Thehomogenate is centrifuged at 10,000×g for 15 minutes, yielding afloating fat pad and a pellet, which are both discarded, and asupernatant fraction (S1).

The S1 fraction is centrifuged at 100,000×g for 90 minutes, yielding asupernatant fraction and a pellet (P2). The P2 pellet, which containssubcellular membranes, is resuspended in approximately 30 ml of ModifiedExtraction Buffer, and centrifuged again at 100,000×g for 90 minutes.The resulting pellet (P3) is resuspended in approximately 2 ml ModifiedP2 Buffer (100 mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 5% glycerol (w/v),10 mM EDTA). The suspension is then divided into aliquots, frozen inliquid nitrogen and stored at -70° C. for future use.

C. Rapeseed LPAAT

A P2 membrane homogenate from immature embryos of developing rapeseed(Brassica napus) seeds is prepared essentially as described above,except as noted below. Immature Brassica seeds are harvested from plantsgrown in growth chambers and greenhouses. The embryos are dissected fromthe immature seeds and frozen in liquid nitrogen. Approximately 1.66 gof Brassica embryos are ground in 8 ml Modified Extraction Buffer usinga chilled mortar and pestle. Since little starting tissue is used, thehomogenate is not filtered through cheesecloth, but is centrifuged at10,000×g for 50 minutes. The supernatant fraction (S1) is thencentrifuged at 100,000×g for 2 hours, and the resultingmembrane-containing P2 pellet is resuspended in 0.25 ml Modified P2Buffer, frozen in liquid nitrogen, and stored at -70° C. for future use.

Example 3

Characterization of LPAAT Activity in Cell-free Homogenates and P2Membrane Preparations

A. Enzyme activity

Coconut, bay, and rapeseed cell-free homogenates and P2 membranepreparations all display LPAAT activity as measured by the assaydescribed in Example 1A. LPAAT activity is dependent on assay incubationtime and varies with the concentrations of substrates and P2preparation, as expected for enzyme catalysis. Confirmation of theidentity of the reaction product as PA can be obtained by incubating theproduct with phospholipase A2 (available commercially, e.g. purifiedfrom Crotalus atrox venom). Radioactivity is converted to a form whichmigrates on TLC as free fatty acid. As phospholipase A2 removes thefatty acyl group at the sn-2 hydroxyl substituent of PA, this result isconsistent with the radioactive LPAAT product being PA radiolabeled atthe sn-2 position.

B. Substrate specificity

The LPAAT activity involved in triacylglycerol (seed oil) biosynthesisis associated with the cytoplasmic endoplasmic reticulum membranes(sometimes referred to as "microsomes") and prefers acyl-CoAs overacyl-ACPs as donor substrates. A functionally analogous enzyme which isable to utilize both acyl-ACP and acyl-CoA substrates is present inplant plastids (Harwood, in Crit. Rev. Plant Sci. (1989), vol. 8, pp.1-43). The coconut P2 preparation will not utilize 12:0-ACP as the LPAATdonor substrate instead of 12:0-CoA. This indicates that the coconut P2preparation contains the cytoplasmic type of LPAAT appropriate to seedoil biosynthesis. The same assay shows that the 12:0-ACP is nothydrolyzed by the P2 preparation, which demonstrates that the lack of12:0-ACP utilization by coconut LPAAT is not a result of depletion of12:0-ACP by hydrolysis. Similarly, the bay P2 preparation will notsignificantly utilize 18:1-ACP as the LPAAT donor substrate instead of18:1-CoA. Thus, the bay P2 preparation also contains the endoplasmicreticulum type of LPAAT appropriate to seed oil biosynthesis.

Lysophosphatidylcholine (LPC) acyltransferase (LPCAT) is an enzymeanalogous to LPAAT, involved in the biosynthesis of membrane lipids(phosphatidyl choline and derivatives thereof) instead of storage oil.The possibility that the activity measured in the LPAAT assay is nottrue LPAAT, but rather an inefficient action of LPCAT on the LPAATsubstrates, can be tested by direct assay for LPCAT. For example, theLPAAT activity of the coconut P2 preparation with the substratecombination 12:0-CoA+12:0-LPA is readily measurable, whereas the LPCATactivity of the same preparation with the substrates 12:0-CoA+12:0-LPCis undetectable. This indicates that the measured medium-chain LPAATactivity is due to an LPAAT enzyme, and not due to an inefficient,side-reaction of LPCAT. When the substrates all have 18:1 acyl groupsthe activities in the LPAAT and LPCAT assays (coconut or bay P2preparations) are of comparable magnitude. The activities on long-chainsubstrates may represent either a single acyltransferase enzyme able touse LPA and LPC acceptor substrates, or discrete "long-chain" LPAAT andLPCAT enzymes which are present together.

C. Chain-length Specificity

The LPAAT activities of the P2 membrane preparations are furthercharacterized with respect to chain-length preference for the donor andacceptor substrates. The following table presents results of LPAATactivity analysis of P2 membrane preparations from coconut, bay, andrapeseed. LPAAT activity is measured with using a variety of acyl-CoAdonor substrates, with the acceptor substrate held constant as 12:0-LPA.

    ______________________________________                                        Donor (Acyl-CoA)                                                                           LPAAT Activity* from:                                            Substrate    Coconut     Bay    Rapeseed                                      ______________________________________                                         6:0          3          1       0                                             8:0          6          13      2                                            10:0         43          10     12                                            12:0         238         14     79                                            14:0         61          5      16                                            16:0         21          6      27                                            18:0         13          6      21                                            18:1          9          5      218                                           ______________________________________                                         (*pmol PA formed/30 min assay)                                           

The coconut LPAAT activity demonstrates a dramatic preference for12:0-containing donor substrate, and also readily utilizes additionalmedium-chain donor acyl-CoA substrates (10:0- and 14:0-containingacyl-CoA substrates). The bay LPAAT activity when 12:0-LPA is theacceptor substrate demonstrates a preference for medium-chain acyl-CoAsubstrates (8:0-, 10:0- and 12:0-containing). Rapeseed LPAAT prefers the18:1 donor when 12:0-LPA is the acceptor, in agreement with previouscharacterizations.

Similar acyl-CoA preferences are observed when assaying coconut LPAATactivity with 18:1-LPA as the acceptor substrate. However, due todifferences in substrate kinetics for 12:0-LPA and 18:1-LPA, directcomparisons of LPAAT activity on different acceptor substrates using asingle acyl-CoA donor substrate are difficult to make.

In the examples which follow, "medium-chain" LPAAT refers to activityassayed with 12:0-CoA and 12:0-LPA substrates, and "long-chain" LPAATrefers to activity assayed with 18:1-CoA and 18:1-LPA substrates.

D. Other Properties

Using the bay P2 membrane preparation, many detergents are found to beinhibitory when included in the assay. For example, a long-chain LPAATactivity (18:1-CoA and 18:1-LPA as substrates) in bay P2 preparations isinhibited completely by 0.1% (all concentrations quoted as w/v) octylglucoside, 0.002% SDS (sodium dodecyl sulfate), 0.005% Zwittergent 3-14(Calbiochem), 1% Tween 20 or Brij 35, 0.03% Triton X100, and by 0.1%sodium deoxycholate. Exposure of the P2 preparation to higherconcentrations than these is possible without permanent loss of enzymeactivity, provided the enzyme-plus-detergent mixture is diluted prior toassay to reduce the detergent concentration to a level which istolerated. For example, the bay P2 preparation can be subjected to a1-hour exposure to 1.25% Brij 35, 0.5% octyl glucoside, 0.1% TritonX-100, or 2.5% Tween 20 without complete loss of activity, provided thepreparation is diluted prior to assay to reduce these detergentconcentrations (to 0.025, 0.01, 0.002, and 0.05% respectively).

The detergent CHAPS, used for solubilization as described in theexamples which follow, is inhibitory in the coconut medium-chain LPAATassay at concentrations above 0.1% (w/v). Accordingly CHAPS-solubilizedLPAAT must be assayed after dilution to reduce the CHAPS concentrationto 0.1% or less. Prior exposure of the coconut P2 preparation to higherCHAPS concentrations, such as 0.5% (w/v), is possible with only partialLPAAT activity loss (50% in this example), provided the dilution isundertaken prior to assay. This phenomenon of tolerance of higherdetergent concentrations than can be accepted in the assay provides abasis for screening for solubilization conditions.

The coconut, P2, medium-chain LPAAT activity is unaffected by 0.1 mMCoA, 2 mM adenosine-5'-triphosphate, or 60 μM lysophosphatidylcholine inthe assay system.

The long-chain LPAAT activity of the bay P2 preparation varies with pHin the assay, being detectable between pH 6 and 10, high between pH 7and 9, and maximal at pH 8. The medium-chain LPAAT activity of thecoconut P2 preparation also shows little change when the assay is rangedbetween pH 6.5 and 8.5 (in 0.5 pH increments), and there is a slightpreference for pH 8.0.

Example 4

Solubilization of LPAAT Activity

A. Coconut Medium-chain and Bay Long-chain LPAATs

All steps are carried out at 0-4° C. The frozen coconut P2 preparationis thawed and diluted in a volume of P2 Buffer to achieve a proteinconcentration of 0.94 mg/ml P2 protein. Protein concentration isdetermined by Coomassie dye staining relative to a bovine serum albuminstandard. The P2 membrane suspension is then diluted with an equalvolume of Solubilization Buffer (50 mM HEPES-NaOH, pH7.5, 1.8M NaCl, 20%(w/v) glycerol, 4.5% (w/v) CHAPS, 100 μM Pefabloc, 1 μM leupeptin, 1 μMPepstatin A, and 5 mM β-ME), resulting in final concentrations of 1MNaCl, 2.25% (w/v) detergent, and 0.47 mg/ml protein. These componentconcentrations, and the resulting detergent/protein ratio of 48/1 (w/w),are important for optimal solubilization. The preparation is thenincubated on ice for 30 minutes with occasional, gentle stirring,followed by centrifugation at 252,000×g for 2 hours. The resultingsupernatant fraction (S3) is filtered through buffer-wetted Miracloth,and may then be stored frozen (-70) with only slight loss of activity.Optimally, it is applied to chromatography columns without anintervening freeze-thaw cycle.

The bay long-chain LPAAT activity in the bay P2 membrane sample issolubilized in the same manner, with the Solubilization Buffer CHAPS andNaCl concentrations being 4% (w/v) and 1M respectively, and thedetergent/protein ratio being 58/1 (w/w).

The detergent BIGCHAP (N,N-bis 3-D-gluconamidopropyl!-cholamide) mayalso be substituted for CHAPS in solubilization of either bay or coconutLPAAT, provided the BIGCHAP concentration in the final mixture is 4%(w/v) and a larger portion of the P2 preparation is used so that thedetergent/protein ratio is unchanged.

B. Evidence for Solubilization

"Solubilization" refers to extraction of the LPAAT enzyme from themembranes present in the P2 preparation, in such a way that it thenbehaves in a manner typical of enzymes that are not membrane-associated.In testing for solubilization of LPAAT activity, the followingindications of solubilization are considered:

1) LPAAT activity is not sedimented by high-speed centrifugationequivalent to, or of larger, g force than that used to sediment the P2membranes.

2) LPAAT activity migrates on a size-exclusion chromatography column asthough it had a native molecular weight typical of enzymes which are notmembrane-associated.

3) Proteins present in the LPAAT preparation will be at least partiallyseparable from each other by column chromatography.

Preparation of the coconut and bay S3 sample having LPAAT activityinvolves centrifugation at much greater g force (252,000×g) than wasused to prepare the original P2 material (100,000×g). A substantialproportion (up to 79%) of the LPAAT activity is found in the resultingsupernatant fraction (S3 preparation), thereby satisfying the firstindication of solubilization.

FIGS. 2-4 show size-exclusion chromatography of the bay long-chain LPAATactivity, using on-column conditions appropriate to the composition ofthe LPAAT preparation being applied. As shown in the first graph (FIG.2), the LPAAT activity of the bay P2 preparation passes through aSephacryl S400 size-exclusion column in the manner of a solute havingextremely high molecular weight. The use of high-molecular-weight dye tocalibrate the column (peak fraction indicated by dotted line labeled"Blue dextran") indicates that the P2 LPAAT activity migrates withoutpenetration into the porous beads of the column, i.e. in the "excluded"or "void" volume. This is typical of enzyme activities associated withmembrane fragments. The second graph (FIG. 3) shows the Sephacryl S400behavior of bay long-chain LPAAT which is prepared from P2 materialaccording to the "solubilization" procedure for pea shoot LPAAT,published by Hares and Frentzen (Planta (1991) 185:124-131). Thisprocedure solubilizes the bay embryo LPAAT according to the firstindication based on centrifugation. However, it does not lead tosignificant LPAAT activity which chromatographs as a protein of lowmolecular weight on a size-exclusion column. Most of the activitycontinues to elute from the column with very high molecular weightcharacteristic of membrane fragments. This observation serves toillustrate that the centrifugation criterion alone is insufficientevidence for solubilization.

In contrast, the LPAAT activity of the bay S3 preparation migrates moreslowly through a size-exclusion column and emerges after a larger volumeof buffer has passed through, as shown in FIG. 4. (In the example showna Superose 6 column is used, to enable finer resolution of proteins inthe 12-200 kDa range). This behavior is typical of enzymes where theprotein molecules are in free solution, not associated with membranefragments. From the elution volumes of various enzymes used for testpurposes (indicated by dotted lines on the graph) it is possible tocalibrate the column, and to conclude that the LPAAT activity of the S3preparation behaves as though it is a globular protein with anapproximate molecular weight of 80 kDa. Since most enzymes which are notassociated with membranes possess molecular weights in the range 20-100kDa, this "apparent molecular weight" is consistent with the conclusionthat the LPAAT has been solubilized. Closely similar results areobtained with the coconut S3 preparation (assaying medium-chainactivity), except that the apparent molecular weight is estimated as44-50 kDa.

Examination of the protein composition of effluent fractions from suchsize-exclusion chromatography of the coconut preparation, by SDS-PAGE(polyacrylamide gel electrophoresis), shows that many proteins arepresent. But the composition varies as fractions are examined from oneend of the LPAAT activity peak to the other. Such protein fractionationwould not be possible if the P2 membranes had not been dispersed intotheir individual lipid and protein constituents, i.e. solubilized.Additional evidence of protein resolution is obtained from applicationof other types of chromatography to the S3 preparation, as in theexamples which follow in the section on purification. Furthermore, bymeans of additional chromatography it is possible to recognizeindividual proteins as candidate proteins for the LPAAT enzyme. Thisobservation provides evidence that the LPAAT protein itself is amongstthose which are dissociated from the membrane in the solubilizationprocedure.

C. Properties of Solubilized Coconut LPAAT

Varying the CHAPS and NaCl concentrations, and the detergent/proteinratio (D/P, w/w), of the solubilizaton procedure results in varyingdegrees of conversion of coconut medium-chain LPAAT activity from the P2preparation to the S3 preparation (i.e. on solubilization as defined bythe centrifugation criterion). FIG. 5 summarizes the effects of CHAPSconcentration (at 1M NaCl) and detergent/protein ratio (D/P, w/w).Lowering the solubilization NaCl concentration below 1 M reduces theformation of S3 LPAAT activity (data not shown in figure). The routinesolubilization conditions are chosen by selecting the minimum CHAPSconcentration for maximal effect (2.25% w/v), and the most effective D/Pratio (48/1 w/w).

Re-examination of the substrate specificity shows that aftersolubilization and phospholipid-activation coconut LPAAT (S3preparation) has the same preference for medium-chain acyl-CoAs as theoriginal P2 activity. Also preserved is the comparable use of 12:0-LPAand 18:1-LPA as acceptor substrates. Assay of the coconut medium-chainLPAAT activity after solubilization (S3 preparation) and reactivationwith PLs, using different acyl-CoA substrates, provides the followingresults. In all these assays the acceptor substrate is 12:0-LPA.

    ______________________________________                                        Acyl-CoA     LPAAT Activity*                                                  ______________________________________                                         6:0          1                                                                8:0         16                                                               10:0         162                                                              12:0         205                                                              14:0         84                                                               16:0         18                                                               18:1         30                                                               ______________________________________                                         *Radioactivity (cpm) of PA product resolved on TLC, after 30 min assay.  

Comparing these results with the P2 membrane activities, it is seen thatthe PL-reactivated, solubilized (S3) activity retains the preference formedium-chain acyl-CoAs.

Increasing the EDTA concentration to 10 mM does not affect the LPAATactivity of the coconut S3 preparation. The additions of 1 mM Mg²⁺,Mn²⁺, or Ca²⁺ are also without significant effect, but the activity isreduced by 50% or more if these ions are added at 10 mM. Omitting β-MEfrom the assay system results in approximately 50% less LPAAT activity,and concentrations above 5 mM also reduce activity. Lowering the assaypH from 7.7 to 6.5 results in a loss of approximately 20% of the LPAATactivity. Raising the pH to 8.0 results in a very slight increase ofactivity which diminishes again as the pH is raised further to 8.5. Theoptimum pH is therefore 8.0, but 7.5 is used routinely to minimizenonenzymatic hydrolysis of acyl-CoAs. There is little change in theactivity when the assay concentration of NaCl is varied between 100 mMand 200 mM, but activity declines steeply as the NaCl concentration israised above 200 mM. Activity is insensitive to changes in glycerolconcentration in the assay between 5% and 15% (w/v).

Overnight dialysis of the coconut S3 preparation to remove NaCl resultsin loss of half of the LPAAT activity. The equivalent NaCl removal usinga size-exclusion column results in total activity loss. Stability of thecoconut S3 preparation during storage at 4° C. is considerably improvedonce it has been activated with phospholipids.

Example 5

Purification of Coconut Medium-Chain LPAAT

Substantial purification of LPAAT activity relative to the total proteincontent of the coconut S3 preparation can be obtained by sequentialchromatography on columns of red 120 agarose and hydroxyapatite, asfollows. The following steps are conducted at 0°-4° C. for optimalrecovery of LPAAT activity.

A. Red 120 Agarose Chromatography

The S3 preparation is diluted to reduce the CHAPS concentration to1.125% (w/v) and the NaCl concentration to 0.5M, all other conditionsremaining the same. It is then applied at 0.5 ml/min to a 2.5 cm(diam.)×2 cm column of red 120 agarose (Sigma Chemical Co., St. Louis)pre-equilibrated in running buffer containing 50 mM HEPES-NaOH, pH 7.5,20% (w/v) glycerol, 1% (w/v) CHAPS, 0.5M NaCl, 5 mM β-ME. Fractions of 3ml volume are collected. As shown in FIG. 6, LPAAT activity is retainedby the column while considerable non-LPAAT protein (assayed by theCoomassie dye method) flows through.

The LPAAT activity is eluted by applying running buffer in which theNaCl concentration is adjusted to 2.5M. A sharp peak of proteinaccompanies the eluted activity. The LPAAT activity recovery from thisprocedure is typically close to 100%, and typically 85% of the proteinsin the coconut LPAAT S3 preparation are removed.

B. Hydroxylapatite Chromatography

The LPAAT-active fractions from the red column, in the buffer containing2.5M NaCl, are pooled and applied to a 1.5 cm (diam.)×5.7 cm HA(hydroxylapatite) column pre-equilibrated with running buffer containing50 mM HEPES-NaOH, pH 7.5, 20% (w/v) glycerol, 1% (w/v) CHAPS, 1M NaCl, 5mM β-ME. The flow rate is again 0.5 ml/min and fractions of 2 ml volumeare collected. Essentially all of the protein and the LPAAT activity inthe sample are bound by the column. The LPAAT activity and bound proteinare substantially resolved by elution with a linear, 0-100 mM phosphateconcentration gradient in the running buffer. These results areillustrated in FIG. 7.

The recovery of activity on this column is typically 60-70%. TheLPAAT-active fractions are pooled and stored at -70° C. after freezingin liquid nitrogen. This active pool forms the starting material foradditional purification experiments. Analysis of this preparation bysize-exclusion chromatography shows that the LPAAT activity stillbehaves as though it were a globular protein of apparent molecularweight 44-50 kDa. This indicates that the partial purification throughthe red and HA columns does not result in any significant aggregation ofthe LPAAT with itself or with other proteins in the preparation, anddoes not compromise the solubilized state of the LPAAT protein.

In a typical application of this 2-column procedure, the final coconutLPAAT preparation contains 17% of the S1 activity and only 0.4% of theS1 protein. This represents a 40-fold purification of LPAAT relative tothe S1 preparation.

Coconut LPAAT activity from the red+HA column sequence still prefers12:0-CoA over 18:1-CoA as donor substrate, and will still utilize12:0-LPA and 18:1-LPA as acceptor substrates. It still decreases as theassay NaCl concentration is raised above 200 mM, and tolerates freezingand thawing with minimal loss.

Example 6

Identification of Coconut LPAAT Protein

A. SDS PAGE Analysis of LPAAT from Hydroxylapatite Column

The protein composition of the LPAAT preparation obtained from the HAcolumn is analyzed by SDS-PAGE. Visualization of the protein compositionof P2, S3, or partially purified S3 preparations by SDS-PAGE requiresthat the sample not be boiled in the SDS-containing PAGE sample bufferprior to loading the gel. SDS-PAGE analysis reveals the presence ofnumerous protein species in the enriched LPAAT preparation. Although theprotein composition is simplified relative to that of the S1preparation, additional chromatography is required to identify theprotein (or proteins) corresponding to LPAAT activity.

B. LPAAT Chromatography on 12:0-CoA Matrix

Useful resolution of the remaining proteins is obtained bychromatography on a matrix comprising immobilized 12:0-CoA substrate(unlabeled). The column matrix is prepared by attaching the amino groupof the CoA moiety of 12:0-CoA to the free carboxyl group of6-aminohexanoic acid Sepharose 4B. This Sepharose derivative, couplingprocedure, and other necessary reagents are obtained from Sigma ChemicalCompany (St. Louis). A density of coupled 12:0-CoA of 3.9mg/ml wet beadvolume can be achieved. A 1 cm-diameter column is prepared with 2 ml ofthe 12:0-CoA matrix, and equilibrated with running buffer containing 50mM HEPES-NaOH pH 7.5, 20% (w/v) glycerol, 1% (w/v) CHAPS, 0.4M NaCl, 5mM β-ME at 0.2-0.5 ml/min.

The LPAAT preparation prepared by chromatography from the red and HAcolumns is diluted with running buffer lacking NaCl, lowering the NaClconcentration to 0.4M, and applied to the 12:0-CoA column. Fractions of2 ml volume are collected. As shown in FIG. 8, a small amount of LPAATactivity emerges during the loading stage. However, the majority of theLPAAT activity is bound to the column and can be eluted later byapplication of a linear 0.4-2M NaCl gradient in the running buffer.Typically 50-60% of the loaded activity is recovered in this NaCl-elutedpeak. If the experiment is repeated with the 6-aminohexanoic acidSepharose 4B support lacking 12:0-CoA, most of the activity emerges inthe loading effluent.

C. SDS PAGE Analysis of LPAAT from 12:0-CoA Column

Analysis of fractions eluted from the 12:0-CoA column by SDS-PAGE andsilver-staining shows that considerable resolution of proteins isaccomplished. Loading and washing fractions 7 and 10 (FIG. 8) contain acomplex protein composition comparable to the sample loaded. Salt-elutedfractions 29-36 (FIG. 8) contain a much simpler protein composition asshown by two prominent component bands and 6-7 less abundant ones.Several very minor components are also detectable in this sample. Theprotein composition of such material varies somewhat from one coconutpreparation to another, but the considerable purification obtained withthe 12:0-CoA column is reproducible. Furthermore, on theSDS-polyacrylamide gel, a band or pair of bands corresponding toproteins having an approximate molecular weight of 27-29 kDa (i.e.migrating slightly faster in the gel than a marker protein of 31 kDa) ismost prominent in intensity in fractions 32 and 33. These fractions alsocontain the maximum LPAAT activity. The 27-29 kDa band consistentlytracks with LPAAT activity in the various coconut 12:0-CoA columnsamples examined. This is strong evidence that the 27-29 kDa protein(also referred to hereafter as the "29 kDa" protein or candidateprotein) corresponds to the LPAAT enzyme. The other proteins infractions 29-36 are most abundant in those fractions which are not atthe peak of LPAAT activity, and are therefore less likely to representLPAAT.

D. Chromatography of Activated LPAAT on 12:0-CoA Matrix

In a modification of the above 12:0-CoA chromatography method, LPAAT isactivated by addition of phospholipids prior to loading on the column.In addition, the running buffer is modified to include phospholipids. Bythese modifications, the LPAAT is maintained in activated formthroughout the experiment.

To prepare modified running buffer, 380 μl of a detergent solution ofphospholipids (50 mg/ml in 0.5% (w/v) CHAPS as described for themodified assay) are mixed with 9.5 ml of HA column running buffer andthis mixture is then diluted by addition of 90 ml CHAPS-free buffercomprising 50 mM HEPES-NaOH, pH 7.5, 20% (w/v) glycerol, 0.44M NaCl, 5mM β-ME. This results in final CHAPS and NaCl concentrations of 0.1%(w/v) and 0.5M respectively, and a phospholipid concentration asdescribed for assay of solubilized LPAAT. Enzyme dilution buffer isprepared with phospholipids in the same manner, but such that the finalCHAPS and NaCl concentrations are 0.1% (w/v) and 0.46M respectively.This dilution buffer is used to dilute the LPAAT sample from the HAcolumn tenfold prior to loading on the 12:0-CoA column.

When applied in the presence of phospholipids only a small amount ofLPAAT activity fails to be retained by the column. The activity may thenbe eluted at a slow rate as the column is washed with running buffer(FIG. 9). Application of 15 ml of 0.1 mM 12:0-LPA in the running bufferresults in the elution of a single large peak of LPAAT activity.Subsequent application of 2.5M NaCl fails to elute additional detectableLPAAT.

Attempts to elute LPAAT from the 12:0-CoA column with 12:0-LPA or18:1-LPA are unsuccessful (or provide only a very small peak ofactivity) unless the LPAAT is activated with phospholipids beforeloading and the column is run with phospholipid-containing buffer in themanner just described. This suggests that LPAAT binds differently to thecolumn when it has been activated with phospholipids, and that thisbinding is based on recognition of the 12:0-CoA moiety of the column bythe catalytic site of the LPAAT protein. The 12:0-LPA elution would thenderive from recognition of the 12:0-LPA substrate by the LPAAT catalyticsite also. These binding and elution phenomena, if based on thecatalytic site, would be expected to be specific for LPAAT and to offerthe prospect of considerable purification.

E. SDS PAGE Analysis of LPAAT from Activated 12:0-CoA Column

Examination of the eluted fractions by SDS-PAGE (with silver staining)shows that different proteins are present in the loading effluent, theLPAAT-active fractions, and the 2.5M NaCl effluent. The significantlystained 29 kDa LPAAT candidate protein is seen in the LPAAT-activefractions, along with several weakly staining protein bands. The 29 kDaprotein is not detected in the LPAAT-inactive fractions. These resultsprovide additional evidence that the 29 kDa protein represents coconutLPAAT.

F. Additional Chromatographic Analyses

Many other chromatography columns may be tested for their ability toresolve proteins present in active LPAAT preparations from the red+HAcolumn sequence. Columns that are useful in this respect includePharmacia "Mono Q" anion exchanger, Merck thiophilic agarose, sizeexclusion columns, and blue 4 agarose. In all these chromatographicanalyses, LPAAT activity can be retained by the column and eluted invarious ways, always accompanied by a protein or pair of proteins havingan apparent molecular weight on SDS-PAGE of approximately 29 kDa.

Thus, the chromatographic evidence demonstrates the relationship betweenLPAAT activity and the protein or proteins migrating with apparentmolecular weight of approximately 29 kDa on SDS-PAGE. Although thismolecular weight does not correspond to the estimate of 44-50 kDa forthe native enzyme obtained by size-exclusion chromatography, suchdifferences between the molecular weights of denatured proteins onSDS-PAGE and the corresponding proteins in the native state are common.These differences can result from the association of the proteinmolecules into diners, tetramers etc. in the native situation, or thebinding of limited numbers of detergent molecules etc. duringsolubilization.

Example 7

Determination of LPAAT Amino Acid Sequence

A. Transfer of LPAAT to Membranes

LPAAT may be further purified for use in determination of amino acidsequence by transfer of the LPAAT preparation resulting from the Red 120and HA column chromatography purification to nitrocellulose or PVDFmembranes following SDS-PAGE. For example, for further use in trypticdigestions, the LPAAT protein is transferred to nitrocellulose. PVDFmembranes, such as ProBlott (Applied Biosystems; Foster City, Calif.)and Immobilon-P (Millipore; Bedford, Mass.) find preferential use indifferent methods. For example, transfer to ProBlott is useful forN-terminal sequencing methods. For generation of peptides from cyanogenbromide digestion, Immobilon-P is preferred.

1. Blotting to Nitrocellulose: When protein is electroblotted tonitrocellulose, the blotting time is typically 1-5 hours in a buffersuch as 25 mM Tris (tris(hydroxymethyl)aminomethane), 192 mM glycine in5-20% methanol. Following electroblotting, membranes are stained in 0.1%(w/v) Ponceau S in 1% (v/v) acetic acid for 2 minutes and destained in2-3 changes of 0.1% (v/v) acetic acid, 2 minutes for each change. Thesemembranes are then stored wet in heat-sealed plastic bags at -20° C. Iftime permits, blots are not frozen but used immediately for digestion tocreate peptides for determination of amino acid sequence as describedbelow.

2. Blotting to PVDF: When protein is electroblotted to Immobilon P PVDF,the blotting time is generally about 1-2 hours in a buffer such as 25 mMTris/192 mM glycine in 20% (v/v) methanol. Following electroblotting toPVDF, membranes are stained in 0.1% (w/v) Coomassie Blue in 50% (v/v)methanol/10% (v/v) acetic acid for 5 minutes and destained in 2-3changes of 50% (v/v) methanol/10% (v/v) acetic acid, 2 minutes for eachchange. PVDF membranes are then allowed to air dry for 30 minutes andare then stored dry in heat-sealed plastic bags at -20° C. Proteinblotted to PVDF membranes such as Pro Blott, may be used directly todetermine N-terminal sequence of the intact protein. A protocol forelectroblotting proteins to ProBlott is described below.

B. Protease Digestion and Separation of Peptides

LPAAT protein that has been blotted to nitrocellulose may be subjectedto digestion with proteases in order to obtain peptides for sequencing.The method used is that of Aebersold, et al. (PNAS (1987) 84:6970).

The LPAAT preparation is transferred to nitrocellulose as describedabove. The band representing the above-identified 29 kDa protein, andalso an equal amount of blank nitrocellulose to be used as a control,are cut out of the nitrocellulose membrane. A 1.0 ml aliquot of 0.5%polyvinylpyrrolidone (PVP-40, Aldrich, Milwaukee, Wis.) in 100 mM aceticacid is added to the membrane pieces and the mixture incubated for 30minutes at 37° C. In order to remove the PVP-40 completely,nitrocellulose pieces are washed with HPLC grade water (6×3 ml),checking the absorbance of the washes at 214 nm on a spectrophotometer.PVP-40 may be more easily removed if bands are not cut into small piecesuntil after PVP-40 treatment and washing.

Following the PVP-40 treatment, the membrane pieces are minced intosmall chips (˜1 mm×1 mm) prior to digestion. The protein is thensuspended in trypsin digest buffer (100 mM sodium bicarbonate pH 8.2).Acetonitrile is added to the digest mixture to a concentration of 5-10%(v/v). Trypsin is diluted in digest buffer and added to the digestmixture, at a ratio of 1:10 (w/w) protease to protein. Digests areincubated 18-24 hours at 37° C.

Following overnight incubation, the digest reaction is stopped byaddition of 10 μgl of 10% (v/v) trifluoroacetic acid (TFA) or 1 μl 100%TFA. The peptides in the digest mixture are separated on a Vydac reversephase C18 column (2.1 mm×150 mm) installed in an Applied Biosystems(Foster City, Calif.) Model 130 High Performance Liquid Chromatograph(HPLC). Mobile phases used to elute peptides are: Buffer A: 0.1 mMsodium phosphate, pH2.2; Buffer B: 70% acetonitrile in 0.1 mM sodiumphosphate, pH2.2. A 3-step gradient of 10-55% buffer B over two hours,55-75% buffer B over 5 minutes, and 75% buffer B isocratic for 15minutes at a flow rate of 50 ml/minute is used. Peptides are detected at214 nm, collected by hand, and stored at -20° C.

Other proteases may also be used to digest the LPAAT protein inappropriate digest buffers, for example, endoproteinase gluc buffer (25mM ammonium carbonate/1 mM EDTA, pH 7.8), or endoproteinase Asp-N buffer(0.05M sodium bicarbonate pH 8.0). In addition, buffer conditions, suchas temperature may vary, for example endoproteinase gluC digestion isconducted at room temperature. However, the protocols for digestion,peptide separation and purification are substantially as described abovefor digestion with trypsin.

C. Cyanogen Bromide Cleavage and Separation of Peptides

Cyanogen bromide cleavage may be performed on LPAAT protein using themethodology described in the Probe-Design Peptide Separation SystemTechnical Manual from Promega, Inc. (Madison, Wis.). The LPAAT proteinpreparation is blotted to a PVDF membrane as described above. Theportion of the membrane containing the transferred 29 kD band is cutfrom the blot, placed in a solution of cyanogen bromide in 70% (v/v)formic acid, and incubated overnight at room temperature. Following thisincubation the cyanogen bromide solutions are removed, pooled and driedunder a continuous nitrogen stream using a Reacti-Vap Evaporator(Pierce, Rockford, Ill.), or evaporated using a Speed-Vac. Additionalelution of cyanogen bromide peptides from PVDF may be conducted toensure complete removal, using a peptide elution solvent such as 70%(v/v) isopropanol, 0.2% (v/v) trifluoroacetic acid, 0.1 mM lysine, and0.1 mM thioglycolic acid. The elution solvents are then removed andadded to the tube containing the dried cyanogen bromide solution, anddried as described above. The elution procedure may be repeated withfresh elution solvent. 50 μl of HPLC grade water is then added to thedried peptides and the water removed by evaporation in a Speed-Vac(Savant, Inc., Farmingdale, N.Y.).

Peptides generated by cyanogen bromide cleavage are separated using aTris/Tricine SDS-PAGE system similar to that described by Schagger andvon Jagow (Anal. Biochem. (1987) 166:368-379). Gels are run at aconstant voltage of 125-150 volts for approximately 1.5 hours or untilthe tracking dye has begun to run off the bottom edge of the gel. Gelsmay be pre-soaked in transfer buffer (125 mM Tris, 50 mM glycine, 10%(v/v) methanol) for 15-30 minutes prior to transfer. Gels are blotted toProBlott sequencing membranes (Applied Biosystems, Foster City, Calif.)for 2 hours at a constant voltage of 50 volts. The membranes are stainedwith Coomassie blue (0.1% in 50% (v/v) methanol/10% (v/v) acetic acid)and destained for 3×2 min. in 50% (v/v) methanol/10% (v/v) acetic acid.Membranes are air-dried for 30-45 minutes before storing dry at -20° C.

Peptides blotted on to ProBlott can be directly loaded to the sequencercartridge of the protein sequencer without the addition of aPolybrene-coated glass fibre filter. Peptides are sequenced using aslightly modified reaction cycle, BLOT-1, supplied by AppliedBiosystems. Also, solution S3 (butyl chloride), is replaced by a 50:50mix of S1 and S2 (n-heptane and ethyl acetate). These two modificationsare used whenever samples blotted to ProBlott are sequenced.

D. N-terminal Sequencing of Proteins and Peptides

Sequencing is performed by Edman degradation on an Applied Biosystems477A Pulsed-Liquid Phase Protein Sequencer; phenylthiohydantoin (PTH)amino acids produced by the sequencer are analyzed by an on-line AppliedBiosystems 120A PTH Analyzer. Data are collected and stored using anApplied BioSystems model 610A data analysis system for the AppleMacintosh and also on to a Digital Microvax using ACCESS*CHROM softwarefrom PE NELSON, Inc. (Cupertino, Calif.). Sequence data is read from achart recorder, which receives input from the PTH Analyzer, and isconfirmed using quantitative data obtained from the model 610A software.

For peptide samples obtained as peaks off of an HPLC, the sample isloaded on to a Polybrene coated glass fiber filter (Applied Biosystems,Foster City, Calif.) which has been pre-washed. For peptides which havebeen reduced and alkylated, a portion of the PTH-amino acid productmaterial from each sequencer cycle is counted in a liquid scintillationcounter. For protein samples which have been electroblotted toImmobilon-P, the band of interest is cut out and then placed above aPolybrene coated glass fiber filter, pre-washed as above and thereaction cartridge is assembled according to manufacturer'sspecifications. For protein samples which have been electroblotted toProBlott, the glass fiber filter is not required.

In order to obtain protein sequences from small amounts of sample (5-30pmoles), the 477A conversion cycle, the S4_(B) solvent and the 120Aanalyzer program are modified as described by Tempst and Riviere (Anal.Biochem. (1989) 183:290).

Amino acid sequence of peptides generated from the 29 kDa LPAAT bytrypsin digestion as described above are as follows:

SQ1256 (SEQ ID NO:1) NLSLIIFPEGTr

SQ1262 (SEQ ID NO:2) YFSPIK

SQ1282 (SEQ ID NO:3) VRPAPITVK

Amino acid sequence of peptides generated from the 29 kDa LPAAT by AspNdigestion as described above are as follows:

SQ1271 (SEQ ID NO:4) TGTHLa

SQ1272 (SEQ ID NO:5) VEMIHaly

SQ1276 (SEQ ID NO:6) slrvrpapitvk

SQ1281 (SEQ ID NO:7) FSPIKT

The amino acid sequence is represented using the one letter code. Aminoacids represented by lower case letters represent residues which wereidentified with a lesser degree of confidence.

E. Homology of LPAAT Peptide to Acyltransferase Proteins

The amino acid sequence of the LPAAT tryptic peptide SQ1256 describedabove is compared to known protein sequences in a computer data bank bycomputer aided homology search. Significant homology is found betweenthe LPAAT peptide and the LPAAT encoded by the E. coli plsC gene. A sixamino acid stretch of the 12 amino acid coconut LPAAT tryptic peptide isan identical match to amino acids 145-150 of the E. coli LPAAT (Colemanet al., supra). In addition, this same conserved six amino acid sequenceis also found at amino acids 154-159 of a yeast acyltransferase proteinencoded by the SLC1 gene. Additional regions of homology with the E.coli plsC and yeast SLC1 gene products are found in coconut LPAAT aminoacid sequence as determined by translation of nucleic acid sequences ofLPAAT PCR sequences described in Example 9.

Example 8

Preparation of Coconut cDNA Library

A. Total RNA preparation

This procedure is an adaptation of the DNA isolation protocol of Webband Knapp (D. M. Webb and S. J. Knapp, (1990) Plant Molec. Reporter, 8,180-185). The following description assumes the use of 1 g fresh weightof tissue. Frozen immature endosperm tissue (from "green" coconuts asdescribed for LPAAT purification) is powdered by grinding under liquidnitrogen. The powder is added to 10 ml REC buffer (50 mM Tris-HCl, pH 9,0.8M NaCl, 10 mM EDTA, 0.5% w/v CTAB (cetyltrimethyl-ammonium bromide))along with 0.2 g insoluble polyvinylpolypyrrolidone, and ground at roomtemperature. The homogenate is centrifuged for 5 minutes at 12,000×g topellet insoluble material. The resulting supernatant fraction isfiltered through Miracloth into a 3 ml phenol/chloroform preparation(phenol-saturated water/chloroform, 1/1 v/v, set to pH 7 with solid Trisbase). After brief centrifugation as above to facilitate phaseseparation the upper phase is removed and the lower phase discarded. Theupper phase is partitioned again with chloroform, and the top phase isagain recovered.

The RNA is then precipitated by addition of 1 volume ethanol andcollected by brief centrifugation as before. The RNA pellet isredissolved in 1 ml autoclaved 0.05% (w/v) DEPC (diethylpyrocarbonate),and reprecipitated by the addition of 1 ml 4M potassium acetate (pH 5),0.05% (w/v) DEPC and incubation on ice for 2 hours. After collection bybrief centrifugation, the RNA pellet is redissolved in 0.4 ml 0.05%(w/v) DEPC and extracted once more with phenol/chloroform as describedabove. Sufficient 3M potassium acetate (pH 5), 0.05% (w/v) DEPC is addedto make the mixture 0.3M in acetate, followed by addition of two volumesof ethanol to precipitate the RNA. This final RNA precipitate isdissolved in 0.1 ml 0.05% (w/v) DEPC and stored frozen.

B. Construction of cDNA Library

A coconut endosperm cDNA library is constructed using Stratagene's (SanDiego, Calif.) "UniZap" system, with the following modifications to thesynthesis of first-strand cDNA. Forty μg of total RNA from coconutendosperm are reverse-transcribed in a 50 μl reaction volume as follows:The RNA, in H2O, is heated at 65° C. for 20 minutes and chilled on ice.The first-strand synthesis is carried out as recommended by Stratagene,with the substitution of 600U "Superscript" reverse transcriptase,"Superscript" 1st-strand buffer, and DTT, all as supplied by BRL(Bethesda, Md.). The reaction mixture is incubated at 60° C. for 45minutes. The remaining steps in the library synthesis are performed asrecommended in the Stratagene "UniZap" protocol. The unamplified cDNAlibrary obtained by this procedure contains 1.4×10⁶ clones with anaverage insert size of 1.25 kb.

Example 9

Isolation of LPAAT-Encoding Sequences

DNA sequences encoding coconut LPAAT peptides are obtained usingsynthetic oligonucleotides designed from LPAAT peptide sequences. TheLPAAT nucleic acid sequences may be obtained by amplification of DNA bypolymerase chain reaction (PCR) using the oligonucleotides as primers,or alternatively, by screening a cDNA or genomic DNA library byradiolabeling the oligonucleotides for use as probes.

A. Synthetic Oligonucleotides

For use as PCR primers from single stranded DNA templatereverse-transcribed from mRNA, oligonucleotides containing the senseorientation sequence corresponding to LPAAT peptide encoding sequencesare prepared. These oligonucleotides are used as primers for the"forward" amplification reaction to produce sense strand DNA.

For the "reverse" reaction for amplification of the non-coding DNAstrand, an oligonucleotide may be designed to be identical to a portionof a primer used to prepare DNA template for PCR. Alternatively,oligonucleotides which contain sequence complementary to LPAAT peptideencoding sequences may be used in combination with a "forward" LPAAToligonucleotide primer as described above.

Where the LPAAT peptide sequences contain amino acids which may beencoded by a number of different codons, the forward or reverse primersmay be "degenerate" oligonucleotides, i.e. containing a mixture of allor some of the possible encoding sequences for a particular peptideregion. To reduce the number of different oligonucleotides present insuch a mixture, it is preferable to select peptide regions which havethe least number of possible encoding sequences when preparing thesynthetic oligonucleotide for PCR primers. Similarly, where thesynthetic oligonucleotide is to be used to directly screen a library forLPAAT sequences, lower degeneracy oligonucleotides are preferred.

In addition to LPAAT encoding sequence, oligonucleotides for primers inPCR will contain additional, non-LPAAT, sequences to aid in cloning ofthe PCR products into convenient plasmid vectors. The non-LPAATsequences may be for restriction digestion sites which may be used toclone the PCR fragments into various plasmids, or may be designed tocontain sequences useful for cloning into a particular commerciallyavailable vector. For example, the synthetic oligonucleotides describedbelow contain sequences useful for cloning using the CLONEAMP™ system(GIBCO BRL; Gaithersburg, Md.), which utilizes UDG (uracil DNAglycosylase) for directional cloning of PCR products (Nisson et al.(1991) PCR Meth. and Appl. 1:120-123).

Following are sequences of synthetic oligonucleotides which may be usedto obtain LPAAT sequences. The oligonucleotide names reflect theparticular LPAAT peptide fragment numbers as listed in Table 1. Theletter "F" in the oligonucleotide name designates a PCR forward reactionprimer. The letter "R" designates a PCR reverse reaction primer. Theletter "P" designates an oligonucleotide to be radiolabeled for use as aprobe in cDNA or genomic library screening. The underlined portion ofthe PCR primers indicates the LPAAT peptide encoding sequence.

SQ1256-1 5' CUACUACUACUAATHATHTTYCCOGARGG 3'(SEQ ID NO:8)

SQ1256-R1 5' CAUCAUCAUCAUCCYTCOGGRAAIATIAT 3'(SEQ ID NO:9)

SQ1262-F1 5' CUACUACUACUATAYTTYWSOCCOATHAA 3'(SEQ ID NO:10)

SQ1262-R1 5' CAUCAUCAUCAUYTTDATOGGOSWRAARTA 3'(SEQ ID NO:11)

SQ1272-F1 5' CUACUACUACUAGTOGARATGATHCA 3'(SEQ ID NO:12)

SQ1272-R1 5' CAUCAUCAUCAURTGDATCATYTCOAC 3'(SEQ ID NO:13)

SQ1272-P1 5' RTGDATCATYTCOAC 3'(SEQ ID NO:14)

SQ1272-P2 5' RTGDATCATYTCNAC 3'(SEQ ID NO:15)

An oligonucleotide, TSYN, is used for reverse transcription frompoly(A)+ or total RNA to prepare single-stranded DNA for use as a PCRtemplate. In addition to a poly(T) region for binding to the mRNApoly(A) tail, the oligonucleotide contains restriction digestionsequences for HindIII, PstI and SstI. The sequence of TSYN is asfollows:

TSYN 5' CCAAGCTTCTGCAGGAGCTCTTTTTTTTTTTTTTT 3'(SEQ ID NO:16)

An oligonucleotide, 5' RACEAMP, is useful in the reverse reaction of PCRfor amplification of the antisense strand of an LPAAT encoding sequence.It is noted that where the template for PCR is single stranded DNAreverse-transcribed from mRNA, the reverse reaction will not occur untilcompletion of the first forward reaction. The first strand reactionresults in production of a sense strand template which may then be usedin amplification of the antisense DNA strand from the reverse primer. Inaddition to a region of identity with TSYN (restriction digest region),5'RACEAMP contains the 5' CAU stretch used in the CLONEAMP™ cloningsystem. The sequence of 5'RACEAMP™ is as follows:

5'RACEAMP 5' CAUCAUCAUCAUAAGCTTCTGCAGGAGCTC 3'(SEQ ID NO:17)

The nucleotide base codes for the above oligonucleotides are as follows:

A=adenine

C=cytosine

G=guanine

H=adenine, cytosine or thymine

N=adenine, cytosine, guanine or thymine

T=thymine

U=uracil

I=inosine

Y=cytosine or thymine

R=adenine or guanine

O=inosine or cytosine

B. PCR Reactions

Poly(A)+RNA is isolated from total RNA prepared from coconut endospermtissue as described in Example 8. Single-stranded cDNA is prepared fromcoconut poly(A)+ or total RNA by reverse transcription using Superscriptreverse transcriptase (BRL) and TSYN as the oligonucleotide primer. Thereaction is conducted according to manufacturer's directions, exceptthat the reaction is run at 45° C. rather than 37° C.

PCR is conducted in a Perkin Elmer Cetus GeneAmp PCR System 9600 PCRmachine using reverse transcribed single-stranded coconut embryo cDNA astemplate. Commercially available PCR reaction and optimization reagentsare used according to manufacturer's specifications. The followingreactions using the above described synthetic oligonucleotides are run:

    ______________________________________                                        Reaction     Forward Primer                                                                           Reverse Primer                                        ______________________________________                                        1            SQ1256-1   5'RACEAMP                                             2            SQ1262-F1  5'RACEAMP                                             3            SQ1272-F1  5'RACEAMP                                             4            SQ1262-F1  SQ1256-R1                                             5            SQ1262-F1  SQ1272-R1                                             6            SQ1256-1   SQ1262-R1                                             7            SQ1256-1   SQ1272-R1                                             8            SQ1272-F1  SQ1256-R1                                             9            SQ1272-F1  SQ1262-R1                                             ______________________________________                                    

DNA fragments generated in PCR reactions are cloned into pAMP1(CLONEAMP™ system; GIBCO BRL). The DNA sequence of the cloned fragmentsare determined to confirm that the cloned fragments encode LPAATpeptides.

Sequence of two PCR products, 23-2 and 23-4, from reaction 7, and onePCR product, 10-1, from reaction 6, are confirmed as encoding LPAATpeptides by DNA sequence and translated amino acid sequence analysis.The sequences of these reactions are provided in FIGS. 10-12.

In FIG. 10, DNA and translated amino acid sequences of clone 23-2,obtained by PCR with oligonucleotides SQ1256-1 and SQ1272-R1, are shown.Translation of the DNA sequence in portions of two different readingframes is required to locate the expected LPAAT peptide regions encodedin the PCR primers. Translated sequence of nucleotides 13-30 correspondsto amino acids 5-10 of the tryptic peptide SQ1256 (SEQ ID NO:1), whichwere encoded by the forward primer. Nucleotides 245-259 correspond toamino acids 1-5 of the AspN peptide SQ1272 (SEQ ID NO:5), encoded by thereverse primer. Translation of nucleotides 32-259 corresponds toadditional LPAAT peptide sequences. For example, nucleotides 32-37encode amino acids 11-12 of SQ1256, although in a different translationframe from the sequence encoding amino acids 5-10 of SQ1256. From thisinformation, as well as by comparison to sequence of clone 23-4 (FIG.11), it appears that an additional nucleotide not present in LPAATencoding sequence was incorporated into the LPAAT encoding sequence(most likely an extra guanine in nucleotides 27-30) during thepolymerase chain reaction.

In addition to the expected LPAAT amino acid sequences from the forwardand reverse primers, the 23-2 translated sequence corresponds to otherLPAAT peptide sequences. Nucleotides 125-142 encode the AspN peptideSQ1271 (SEQ ID NO:4); nucleotides 155-190 encode the AspN peptide SQ1276(SEQ ID NO:6), as well as tryptic peptide SQ1282 (SEQ ID NO:3) (SQ1282is identical to amino acids 4-12 of SQ1276); and nucleotides 191-211encode the AspN peptide SQ1281 (SEQ ID NO:7) and tryptic peptide SQ1262(SQ ID NO:2).

DNA sequence of a second clone, 23-4, of a larger reaction 7 PCR productis shown in FIG. 11. In this sequence, the last two amino acids of theSQ1256 peptide are encoded in frame with amino acids 5-10 (encoded bythe PCR primer). The difference in size between the 23-4 insert(approximately 360 bp) and the 23-2 product (approximately 270 bp) isapparently due to the presence of an unprocessed intron in the 23-4sequence (untranslated sequence at nucleotides 70-157 of FIG. 11). Thepresence of the intron is likely due to an unprocessed LPAAT RNAA in thetotal RNA (as opposed to poly(A)+) used to generate the single-strandedcDNA PCR template.

Excluding the intron and PCR primer regions, the LPAAT sequences of theinserts in 23-2 and 23-4 match at all but a single nucleotide, namelynucleotide 90 of 23-2, which is a thymine, and corresponding nucleotide177 of 23-4, which is a cytosine. This nucleotide difference alsoresults in a difference in the translated amino acid sequence of 23-2and 23-4. A leucine is encoded by nucleotides 89-91 in 23-2, and aproline is encoded by corresponding nucleotides 176-178 of 23-4.

DNA sequence of the approximately 220 bp insert in the cloned PCRproduct of reaction 6, 10-1, is provided in FIG. 12. The LPAAT encodingsequence of this clone, with the exception of the PCR primer regions, isidentical to that of 23-4 in the shared region.

C. Library Screening

1. Synthetic oligonucleotide as probe: Useful hybridization solutionsfor library screening with oligonucleotide probes, such as SQ1272-P1 orSQ1272-P2, include tetraalkylammonium salt solutions, such as describedby Jacobs, et al. (Nucl. Acids Res. (1988) 16:4637-4650). Appropriatehybridization conditions, such as hybridization and washingtemperatures, may also be determined by Northern analysis of RNA blotscontaining RNA from the enzyme source, i.e. coconut endosperm. Theoligonucleotide may then be radiolabeled and hybridized with clones fromthe coconut cDNA library described above, or from a coconut genomiclibrary, in order to identify clones containing sequences encoding LPAATpeptides.

2. PCR product as probe: LPAAT DNA fragments obtained by PCR asdescribed above may also be radiolabeled and used as probes for coconutLPAAT clones (Maniatis, supra). An approximately 280 bp fragment ofclone 23-2 containing the LPAAT encoding region is obtained by digestionof 23-2 with XbaI and SalI and isolation of the resulting approximately280 bp fragment. The fragment is radiolabeled by random priming using arandom labeling kit (Stratagene; La Jolla, Calif.). Approximately240,000 plaques of the coconut endosperm cDNA library in the UniZapphage are plated, lifted onto nylon membrane filters and hybridized tothe labeled LPAAT 23-2 fragment. Hybridization is conducted at 42° C. inhybridization solution containing 50% formamide, 5×SSC (1×SSC=0.15MNaCl; 0.015M Na citrate), 0.1% SDS, 0.1 mg/ml salmon sperm DNA, 10×Denhardt's solution. The filters are washed in 1× SSC, 0.1% SDS at roomtemperature for 30 minutes, followed by two 30 minute washes in the samesolution at 37° C. A total of 32 hybridizing plaques are identified. Theidentified plaques are replated and hybridization with the radiolabeledplaque is repeated to obtain purified cultures of 30 of the LPAATcontaining phage. The LPAAT cDNA fragments are excised from the UniZapphage vector according to manufacturer's (Stratagene) directions.Briefly, a helper phage system is used which results in automaticexcision and recircularization of excised cDNA to generate subclones ina pBluescript SK- (Stratagene) phagemid vector. The LPAAT subclones arefurther analyzed to determine the lengths of the various inserts and 3'non-coding sequences are obtained and analyzed to determine the numberof classes of LPAAT clones.

Although cDNA clones of various sizes are obtained, DNA sequenceanalysis of the 3' portions of 26 of the clones indicates that they arefrom the same gene. The clones vary in sequence length at both the 5'and the 3' ends. The variation at the 3' ends indicates that more thanone polyadenylation site is used. DNA sequence and translated amino acidsequence of full length clone COLP4 (pCGN5503) is provided in FIG. 13.

The calculated molecular mass of the translated LPAAT protein of COLP4is approximately 34.8 kD, and the estimated isoelectric focusing pointis 9.79. The calculated molecular mass is not inconsistent with theobserved 27-29 kD value from SDS-PAGE.

Two additional clones having the same 5' sequence as COLP4 were alsoexamined. Each of these clones contained a deletion in the LPAATencoding region. In clone COLP25, a 99 bp region (bases 721-819 of FIG.13) is deleted. The proper frame for translation is maintained,resulting in a translated protein lacking a 33 amino acid LPAAT peptideregion. In clone COLP10, a 49 bp region (bases 820-868 of FIG. 13) isdeleted, and the LPAAT reading frame is not maintained.

Example 10

Expression of LPAAT in E. coli

Coconut LPAAT is expressed in E. coli to provide a convenient source ofthe protein for antibody production and for confirmation of expressionof LPAAT activity. The LPAAT cDNA insert from pCGN5503 (COLP4) ismutagenized by PCR to insert a SalI restriction site immediatelyupstream of the ATG start codon at nucleotides 259-261 of the sequenceshown in FIG. 13, and a BamHI site immediately downstream of the TAAstop codon at nucleotides 1183-1185 of the sequence shown in FIG. 13.The LPAAT encoding sequence is cloned as a SalI/BamHI fragment into acommercial cloning vector, CloneAmp (BRL), resulting construct isdesignated pCGN5504.

The LPAAT encoding region in pCGN5504 is transferred as a SalI/BamHIfragment into E. coli expression vector pCGN7645 for expression of LPAATfrom a T7 promoter. pCGN7645 was constructed by cloning a syntheticoligonucleotide linker containing a Shine-Delgarno sequence and SalI,BamHI and PstI restriction sites into XbaI/BamHI digested pET3A(Rosenberg et al. (1987) Gene 56:125-135. The sequence of theoligonucleotide linker is as follows:

5' CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGGTCGACGGATCCCTGCAGATC 3'.

E. coli BL21(DE3) cells containing the LPAAT construct are grown at 37°C. in liquid medium and expression is induced by the addition of 0.4 mMIPTG. Cells are harvested by centrifugation and assayed for LPAATactivity as described in Example 1

Example 11

Constructs for Plant Transformation

DNA constructs for use in plant transformation are prepared. For uses inexpression in plant oilseed crops for modification of TAG, LPAATencoding sequences may be inserted into expression cassettes containingregulatory regions which provide for preferential expression in plantseed tissues. Examples of genes from which such expression cassettes maybe prepared include seed ACP, a Bce4 gene from Brassica seeds, and aBrassica napin gene. See, for example, Kridl et al. (in Control of PlantGene Expression (1993) Chapter 30, pages 481-498, ed. D.P.S. Verma, CRCPress) for a discussion expression cassettes for use in expression ofgenes in plant seed tissues.

A. Napin Expression Construct

A napin expression cassette, pCGN1808, which may be used for expressionof wax synthase or reductase gene constructs is described in Kridl etal. (Seed Science Research (1991) 1:209-219), which is incorporatedherein by reference.

Alternatively, pCGN1808 may be modified to contain flanking restrictionsites to allow movement of only the expression sequences and not theantibiotic resistance marker to binary vectors such as pCGN1557 (McBrideand Summerfelt, supra). Synthetic oligonucleotides containing KpnI, NotIand HindIII restriction sites are annealed and ligated at the uniqueHindIII site of pCGN1808, such that only one HindIII site is recovered.The resulting plasmid, pCGN3200 contains unique HindIII, NotI and KpnIrestriction sites at the 3'-end of the napin 3'-regulatory sequences asconfirmed by sequence analysis.

The majority of the napin expression cassette is subcloned from pCGN3200by digestion with HindIII and SacI and ligation to HindIII and SacIdigested pIC19R (Marsh, et al. (1984) Gene 32:481-485) to make pCGN3212.The extreme 5'-sequences of the napin promoter region are reconstructedby PCR using pCGN3200 as a template and two primers flanking the SacIsite and the junction of the napin 5'-promoter and the pUC backbone ofpCGN3200 from the pCGN1808 construct. The forward primer contains ClaI,HindIII, NotI, and KpnI restriction sites as well as nucleotides 408-423of the napin 5'-sequence (from the EcoRV site) and the reverse primercontains the complement to napin sequences 718-739 which include theunique SacI site in the 5'-promoter. The PCR was performed using aPerkin Elmer/Cetus thermocycler according to manufacturer'sspecifications. The PCR fragment is subcloned as a blunt-ended fragmentinto pUC8 (Vieira and Messing (1982) Gene 19:259-268) and digested withHincII to give pCGN3217. Sequence of pCGN3217 across the napin insertverifies that no improper nucleotides were introduced by PCR. The napin5-sequences in pCGN3217 are ligated to the remainder of the napinexpression cassette by digestion with ClaI and SacI and ligation topCGN3212 digested with ClaI and SacI. The resulting expression cassettepCGN3221, is digested with HindIII and the napin expression sequencesare gel purified away and ligated to pIC20H (Marsh, supra) digested withHindIII. The final expression cassette is pCGN3223, which contains in anampicillin resistant background, essentially identical 1.725 napin 5'and 1.265 3' regulatory sequences as found in pCGN1808. The regulatoryregions are flanked with HindIII, NotI and KpnI restriction sites andunique SalI, BglII, PstI, and XhoI cloning sites are located between the5' and 3' noncoding regions.

The SalI/BamHI fragment of pCGN5504 containing the entire LPAAT encodingregion is ligated into SalI/BglII digested pCGN3223 to provide anexpression construct having the coconut LPAAT encoding sequencepositioned for transcription of the sense sequence under regulation ofthe napin promoter.

B. Oleosin Expression Construct

A cassette for cloning of sequences for transcription under the controlof 5' and 3' regions from an oleosin gene may be prepared as follows.Sequence of a Brassica napus oleosin gene was reported by Lee and Huang(Plant Phys. (1991) 96:1395-1397). Primers to the published sequence areused in PCR reactions to obtain the 5' and 3' regulatory regions of anoleosin gene from Brassica napus cv. Westar. Two PCR reactions wereperformed, one to amplify approximately 950 nucleotides immediatelyupstream of the ATG start codon for the oleosin gene, and one to PCRamplify approximately 600 bp including and downstream of the TAA stopcodon for the oleosin gene. The PCR products were cloned into plasmidvector pAMP1 (BRL) according to manufacturer's protocols to yieldplasmids pCGN7629 which contains the oleosin 5' flanking region andpCGN7630 which contains the 3' flanking region. The PCR primers includedconvenient restriction sites for cloning the 5' and 3' flanking regionstogether into an expression cassette. A PstI fragment containing the 5'flanking region from pCGN7629 was cloned into PstI digested pCGN7630 toyield plasmid pCGN7634. The BssHII (New England BioLabs) fragment frompCGN7634, which contains the entire oleosin expression cassette wascloned into BssHII digested PBCSK+ (Stratagene) to provide the oleosincassette as plasmid pCGN7636. The oleosin cassette is flanked by BssHII,KpnI and XbaI restriction sites, and contains SalI, BamHI and PstI sitesfor insertion of DNA sequences of interest between the 5' and 3' oleosinregions.

The SalI/BamHI fragment of pCGN5504 containing the entire LPAAT encodingregion is ligated into SalI/BamHI digested pCGN7636 to provide anexpression construct having the coconut LPAAT encoding sequencepositioned for transcription of the sense sequence under regulation ofthe oleosin promoter.

C. Binary Constructs for Plant Agrobacterium-Mediated PlantTransformation

Constructs for plant transformation are prepared by transfer of theexpression cassettes containing LPAAT sequences into convenient cloningsites on a binary vector such as those described by McBride et al.(supra). The binary constructs are then transformed into cells of anappropriate Agrobacterium strain, such as EHA101 (Hood et al. (1986) J.Bacteriol. 168:1291-1301) as per the method of Holsters et al. (Mol.Gen. Genet. (1978) 163:181-187) for use in preparation of transgenicplants.

Example 11

Transformation with LPAAT Constructs

A variety of methods have been developed to insert a DNA sequence ofinterest into the genome of a plant host to obtain the transcription ortranscription and translation of the sequence to effect phenotypicchanges.

Transgenic Brassica plants (variety 212/86, for example) are obtained byAgrobacterium-mediated transformation as described by Radke et al.(Theor. Appl. Genet. (1988) 75:685-694; Plant Cell Reports (1992)11:499-505). Transgenic Arabidopsis thaliana plants may be obtained byAgrobacterium-mediated transformation as described by Valverkens et al.,(Proc. Nat. Acad. Sci. (1988) 85:5536-5540). Other plant species may besimilarly transformed using related techniques.

Alternatively, microprojectile bombardment methods, such as described byKlein et al. (Bio/Technology 10:286-291) may also be used to obtainnuclear transformed plants comprising the viral single subunit RNApolymerase expression constructs described herein.

Example 12

Analysis of Transgenic Plants

Seeds from transgenic Brassica plants containing the coconut LPAATconstructs are assayed for C12 LPAAT activity as described in Example 1.Plants identified as positive for LPAAT expression are out-crossed toBrassica plants containing high levels of C12 fatty acids as the resultof expression of a C12 preferring acyl-ACP thioesterase from Californiabay (WO 92/20236 and WO 94/10288). In this manner, a ready source of C12acyl-CoA donor substrate for LPAAT activity is provided.

To identify effects of the expressed LPAAT on the fatty acidcompositions of transgenic seed oils, the fatty acid composition ofextracted oils is determined by acid methanolysis as described by Browseet al. (Anal. Biochem. (1986) 152:141-145). In addition, analysis ofindividual triglyceride types, for example, to determine percentage oftri-laurin triglycerides, may be conducted by HPLC resolution asdescribed by Jeffrey et al. (JAOCS (1991) 68:289-293) orNikolova-Damyanova et al. (JAOCS (1990) 67:503-507).

Analyses of the acyl compositions of the sn-2 and sn-1+3 positions ofTAG are conducted using the pancreatic lipase protocol (Brockerhoff(1975) Meth. Enzymol. 35:315-325). Ideally with this protocol, thelipase cleaves fatty acids from the sn-1 and sn-3 positions, and notfrom the sn-2 position. Thus, the fatty acids in the resultingmono-glyceride are presumed to be those in the sn-2 position. However,it is noted that those previously attempting to study TAG havingshorter-chain fatty acids by this method (Entressangles et al. (1964)Biochim. Biophys. Acta 84:140-148), reported that shorter-chain fattyacids located at the sn-2 position were quickly hydrolyzed during such adigestion, which the authors reported to be the result of a spontaneousmigration of internal shorter-chain fatty acids towards outer positionsin diglycerides and monoglycerides.

Oil distilled from mature seeds is subjected to a pancreatic lipasedigestion protocol modified from Brockerhoff et al., supra, to minimizeacyl migration. This distinguishes acyl compositions of the sn-2 andsn-1+3 combined positions. The modifications are as follows: pH islowered to neutrality, reaction time is shortened from 15 to 3 minutes,samples are maintained at acidic pH thereafter, and digestion productsare chromatographed on borate-impregnated TLC plants. Thechromatographed products are then eluted and analyzed as fatty acidmethyl esters as before. In this manner the percentage of medium-chainfatty acids, and in particular, C12 and C14 fatty acids in the sn-2position is determined.

In the above examples, solubilization and properties of LPAAT activityfrom plant seed tissues are described. A protocol is provided to obtainsubstantially purified medium-chain acyl-CoA-preferring LPAAT fromcoconut endosperm. various properties of the protein are described,including methods to obtain and use amino acid and nucleic acidsequences related thereto. Nucleic acid and amino acid sequencescorresponding to a coconut LPAAT protein are provided, and constructsfor expression of LPAAT in host cells are described. Thus, through thisinvention, one can obtain the amino acid and nucleic acid sequenceswhich encode LPAATs from a variety of sources and for a variety ofapplications. These LPAAT sequences may then be expressed in transgenicplants to obtain altered triacylglycerides as described.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AsnLeuSerLeuIleIlePheProGluGlyThrArg                                          510                                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TyrPheSerProIleLys                                                            (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      ValArgProAlaProIleThrValLys                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      ThrGlyThrHisLeuAla                                                            5                                                                             (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      ValGluMetIleHisAlaLeuTyr                                                      5                                                                             (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      SerLeuArgValArgProAlaProIleThrValLys                                          510                                                                           (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      PheSerProIleLysThr                                                            5                                                                             (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE:N at 24 = inosine or cytosine                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      CUACUACUACUAATHATHTTYCCNGARGG29                                               (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE: N at 18 = inosine or cytosine                                   N at 24 =inosine                                                              N at 27 =inosine                                                              (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      CAUCAUCAUCAUCCYTCNGGRAANATNAT29                                               (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:other                                                      (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE: N at 21 = inosine or cytosine                                   N at 24 =inosine or cytosine                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     CUACUACUACUATAYTTYWSNCCNATHAA29                                               (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE: N at 19 = inosine or cytosine                                   N at 22 =inosine or cytosine                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     CAUCAUCAUCAUYTTDATNGGNSWRAARTA30                                              (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE: N at 15 = inosine or cytosine                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     CUACUACUACUAGTNGARATGATHCA26                                                  (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE: N at 25 = inosine or cytosine                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     CAUCAUCAUCAURTGDATCATYTCNAC27                                                 (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE: N at 13 = inosine or cytosine                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     RTGDATCATYTCNAC15                                                             (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (ix) FEATURE: N at 13 = adenine, cytosine, guanine or thymine                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     RTGDATCATYTCNAC15                                                             (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     CCAAGCTTCTGCAGGAGCTCTTTTTTTTTTTTTTT35                                         (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE: other                                                     (A) DESCRIPTION:synthetic oligonucleotide                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     CAUCAUCAUCAUAAGCTTCTGCAGGAGCTC30                                              (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 271 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     CTACTACTACTAATAATATTTCCGGAGGGGTACTCGATCGAAAACAGGA49                           IleIlePheProGluGlyThrArgSerLysThrGly                                          AGGCTGCTTCCATTTAAGAAGGGTTTTATTCACATAGCACTTCAGACA97                            ArgLeuLeuProPheLysLysGlyPheIleHisIleAlaLeuGlnThr                              CGGTTGCCGATAGTTCCAATGGTGCTGACGGGTACCCATCTAGCTTGG145                           ArgLeuProIleValProMetValLeuThrGlyThrHisLeuAlaTrp                              AGGAAGAACAGTTTGCGAGTCAGACCAGCACCTATCACAGTGAAA190                              ArgLysAsnSerLeuArgValArgProAlaProIleThrValLys                                 TACTTCTCACCCATAAAAACTGATGACTGGGAAGAAGAAAAGATC235                              TyrPheSerProIleLysThrAspAspTrpGluGluGluLysIle                                 AATCATTATGTGGAAATGATCCACATGATGATGATG271                                       AsnHisTyrValGluMetIleHis                                                      (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 358 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     CTACTACTACTAATAATATTCCCCGAAGGTACTCGATCGAAAACAGGAAGG51                         IleIlePheProGluGlyThrArgSerLysThrGlyArg                                       CTGCTTCCATTTAAGAAGGTAACGATCATAACATGCGTGTATATTTGT99                            LeuLeuProPheLysLys                                                            ATGTTTATCCATTTTATTCTTCTGCTTGTGCTTCTCGTTTCTTCATTTTCTGTTGCAG157                 GGTTTTATTCACATAGCACCTCAGACACGGTTGCCGATAGTTCCAATG205                           GlyPheIleHisIleAlaProGlnThrArgLeuProIleValProMet                              GTGCTGACGGGTACCCATCTAGCTTGGAGGAAGAACAGTTTGCGAGTC253                           ValLeuThrGlyThrHisLeuAlaTrpArgLysAsnSerLeuArgVal                              AGACCAGCACCTATCACAGTGAAATACTTCTCACCCATAAAAACTGAT301                           ArgProAlaProIleThrValLysTyrPheSerProIleLysThrAsp                              GACTGGGAAGAAGAAAAGATCAATCATTATGTCGAAATGATTCAC346                              AspTrpGluGluGluLysIleAsnHisTyrValGluMetIleHis                                 ATGATGATGATG358                                                               (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 218 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     TACTACTACTAATAATATTTCCCGAGGGTACTCGATCGAAAACAGGAAGG50                          IleIlePheProGluGlyThrArgSerLysThrGlyArg                                       CTGCTTCCATTTAAGAAGGGTTTTATTCACATAGCACTTCAGACACGG98                            LeuLeuProPheLysLysGlyPheIleHisIleAlaLeuGlnThrArg                              TTGCCGATAGTTCCAATGGTGCTGACGGGTACCCATCTAGCTTGGAGG146                           LeuProIleValProMetValLeuThrGlyThrHisLeuAlaTrpArg                              AAGAACAGTTTGCGAGTCAGACCAGCACCTATCACAGTGAAATACTTT194                           LysAsnSerLeuArgValArgProAlaProIleThrValLysTyrPhe                              TCGCCGATCAAAATGATGATGATG218                                                   SerProIleLys                                                                  (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1408 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     CGGCAGACCCCTCTCTTCTTAGAAACCACCCGTCAGTATTTCTTAATTTTCTTTACTCTT60                TTTCTCTATTTGGTCTGCACTCTAGAATCTTCTCTTTCTTCTCTCTCCACCAAGAACCCA120               TAGAATTTGTTCGTTGCTGGATTCCGATTCCGACCTATTCGCCAGTTCCCTACTCGGAAC180               CCTCAACCCTTTACGTAGTCCTCGTTTGCCTTTCTTGCTCGTGGTATTGGTGGTGGGAAG240               TGGGGGATATATAGTCCTATGGATGCTTCAGGGGCAAGTTCGTTCTTGCGG291                        MetAspAlaSerGlyAlaSerSerPheLeuArg                                             GGCCGTTGTCTGGAGAGCTGCTTCAAAGCGAGCTTCGGGATGTCCCAA339                           GlyArgCysLeuGluSerCysPheLysAlaSerPheGlyMetSerGln                              CCGAAAGATGCAGCCGGGCAACCGAGTCGCCGGCCGGCCGACGCGGAT387                           ProLysAspAlaAlaGlyGlnProSerArgArgProAlaAspAlaAsp                              GACTTTGTGGATGATGATAGATGGATTACTGTCATCCTGTCGGTCGTT435                           AspPheValAspAspAspArgTrpIleThrValIleLeuSerValVal                              AGGATCGCTGCTTGCTTTCTGTCGATGATGGTTACCACCATCGTGTGG483                           ArgIleAlaAlaCysPheLeuSerMetMetValThrThrIleValTrp                              AACATGATCATGCTGATTTTGCTCCCTTGGCCATATGCTCGGATCAGG531                           AsnMetIleMetLeuIleLeuLeuProTrpProTyrAlaArgIleArg                              CAGGGAAACTTGTATGGCCATGTTACCGGGCGGATGCTGATGTGGATC579                           GlnGlyAsnLeuTyrGlyHisValThrGlyArgMetLeuMetTrpIle                              TTAGGGAACCCAATAACAATAGAAGGTTCTGAATTCTCGAACACAAGG627                           LeuGlyAsnProIleThrIleGluGlySerGluPheSerAsnThrArg                              GCCATCTACATCTGTAATCATGCATCACTTGTAGACATTTTTCTCATC675                           AlaIleTyrIleCysAsnHisAlaSerLeuValAspIlePheLeuIle                              ATGTGGTTGATTCCAAAGGGTACCGTTACCATAGCAAAAAAAGAGATC723                           MetTrpLeuIleProLysGlyThrValThrIleAlaLysLysGluIle                              ATTTGGTACCCACTCTTTGGGCAGCTTTATGTATTGGCAAACCATCAG771                           IleTrpTyrProLeuPheGlyGlnLeuTyrValLeuAlaAsnHisGln                              CGAATAGACCGGTCCAACCCATCCGCTGCCATTGAGTCAATTAAAGAG819                           ArgIleAspArgSerAsnProSerAlaAlaIleGluSerIleLysGlu                              GTAGCTCGAGCAGTTGTCAAGAAAAACTTATCGCTGATCATTTTTCCA867                           ValAlaArgAlaValValLysLysAsnLeuSerLeuIleIlePhePro                              GAGGGTACTCGATCGAAAACAGGAAGGCTGCTTCCATTTAAGAAGGGT915                           GluGlyThrArgSerLysThrGlyArgLeuLeuProPheLysLysGly                              TTTATTCACATAGCACTTCAGACACGGTTGCCGATAGTTCCAATGGTG963                           PheIleHisIleAlaLeuGlnThrArgLeuProIleValProMetVal                              CTGACGGGTACCCATCTAGCTTGGAGGAAGAACAGTTTGCGAGTCAGA1011                          LeuThrGlyThrHisLeuAlaTrpArgLysAsnSerLeuArgValArg                              CCAGCACCTATCACAGTGAAATACTTCTCACCCATAAAAACTGATGAC1059                          ProAlaProIleThrValLysTyrPheSerProIleLysThrAspAsp                              TGGGAAGAAGAAAAGATCAATCATTATGTGGAAATGATACATGCCTTG1107                          TrpGluGluGluLysIleAsnHisTyrValGluMetIleHisAlaLeu                              TACGTGGATCACCTGCCGGAGTCTCAAAAACCTTTGGTATCAAAAGGG1155                          TyrValAspHisLeuProGluSerGlnLysProLeuValSerLysGly                              AGGGATGCTAGCGGAAGGTCAAATTCATAAGTATAGGTTTCCTTGAG1202                           ArgAspAlaSerGlyArgSerAsnSer                                                   CATCATGTTGGTTATTATATGCAGCAATATGACAAGCATAAGTGTGACTTATTTTAGAAA1262              TATGTTCATGCCTTTTTTTTTTCCTTATCAGTACCATCATGTGGAATAAAGAAACGCTTT1322              NTGAAAAAAAAAAAAAAAAAAAAAAAAAACTCGAGGGGGGGCCCGGTACCCAATTCGCCC1382              TATAGTGAGTCGTATTACAATCACTG1408                                                __________________________________________________________________________

What is claimed is:
 1. A DNA construct comprising a sequence encodingthe coconut 1-acylglycerol-3-phosphate acyltransferase amino acidsequence shown in any one of FIGS. 10-13 (SEQ ID NO:18-21), wherein saidDNA construct further comprises a DNA sequence not naturally associatedwith said acyltransferase encoding sequence.
 2. A DNA constructcomprising the coconut acyltransferase encoding sequence shown in anyone of FIGS. 10--13 (SEQ ID NOS:18-21).
 3. The construct of claim 2,wherein said plant acyltransferase encoding sequence is from coconutendosperm.
 4. A DNA construct comprising in the 5' to 3' direction oftranscription, a transcriptional initiation region functional in a hostcell, a coconut 1-acylglycerol-3-phosphate acyltransferase proteinencoding sequence, and a transcriptional termination region functionalin said host cell, wherein at least one of said transcriptionalinitiation region or transcriptional termination region is not naturallyassociated with said coconut 1-acylglycerol-3-phosphate acyltransferaseprotein encoding sequence.
 5. The construct of claim 4, wherein saidhost cell is a plant cell.
 6. The construct according to claim 4,wherein said transcriptional initiation region is from a genepreferentially expressed in plant seed tissue.
 7. The construct of claim4, wherein said coconut acyltransferase encoding sequence encodes theacyltransferase protein having the amino acid sequence shown in FIGS.13A-13E (SEQ ID NO:21).
 8. The construct of claim 4, wherein said plant1-acylglycerol-3-phosphate acyltransferase protein is preferentiallyactive towards C8, C10, C12 and C14 acyl-CoA substrates as compared tolonger chain acyl-CoA substrates.
 9. A cell comprising a constructaccording to any one of claims 4 or 6-8.
 10. A plant cell comprising aconstruct according to any one of claim 4 or 6-8.
 11. A plant comprisinga plant cell of claim
 10. 12. A plant of claim 11, wherein said plant isa Brassica plant.
 13. A method of producing a plant1-acylglycerol-3-phosphate acyltransferase in a cellcomprisingtransforming a cell with a DNA construct of claim 4, andgrowing said cell to produce quantities of said plant1-acylglycerol-3-phosphate acyltransferase.